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Periodontitis is a chronic inflammatory disease triggered by oral bacterial infection, with the bacterium Porphyromonas gingivalis being a major causative agent. The association between periodontitis and various systemic diseases has been demonstrated. Recent research has also highlighted the relationship between the aggravation of maternal periodontitis and adverse pregnancy outcomes such as preterm birth and low birth weight. However, the molecular mechanisms underlying how factors from periodontitis influence pregnancy and fetal development remain unclear. Extracellular vesicles (EVs) are nano-sized spherical particles secreted into the tissue microenvironment by various types of cells. EVs have garnered interest in recent years due to their role in intercellular communication. In the present study, we investigated whether EVs derived from P. gingivalis –infected macrophages (Pg-inf EVs) reach the feto-placental unit and influence fetal development. Through a series of in vivo experiments in mice, we demonstrated that Pg-inf EVs translocated to the feto-placental unit and impaired fetal development in size and weight. Histological analysis revealed disoriented blood vessel alignment and impaired angiogenesis in the placentas of Pg-inf EV–injected groups, indicative of compromised placental function. Proteome analysis revealed a significant decrease in Vegfr1 expression in the placentas of the experimental group. Moreover, Pg-inf EVs reduced VEGFR1 expression in cultured human vascular endothelial cells, highlighting a potential molecular mechanism through which these EVs exert their effects on placental angiogenesis. This is the first study to reveal a novel pathway in which oral bacteria–infected macrophage EVs in maternal periodontitis affect pregnancy via the feto-placental unit. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer (PCa) is the second most commonly diagnosed cancer in men worldwide, with metastasis, particularly to bone, being the primary cause of mortality. Currently, prognostic markers like PSA levels and Gleason classification are limited in predicting metastasis, emphasizing the need for novel clinical biomarkers. New molecules predicting tumor progression have been identified over time. Some, such as the immune checkpoint inhibitors (ICIs) PD-1/PD-L1, have become valid markers as theranostic tools essential for prognosis and drug target therapy. However, despite the success of ICIs as an anti-cancer therapy for solid tumors, their efficacy in treating bone metastases has mainly proven ineffective, suggesting intrinsic resistance to this therapy in the bone microenvironment. This study explores the potential of immunological intratumoral biomarkers, focusing on placental growth factor (PlGF), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), and Programmed Cell Death Protein 1 (PD-1), in predicting bone metastasis formation. Methods: we analyzed PCa samples from patients with and without metastasis by immunohistochemical analysis. Results: Results revealed that PlGF expression is significantly higher in primary tumors of patients that developed metastasis within five years from the histological diagnosis. Additionally, PlGF expression correlates with increased VEGFR1 and PD-1 levels, as well as the presence of intratumoral M2 macrophages. Conclusions: These findings suggest that PlGF contributes to an immunosuppressive environment, thus favoring tumor progression and metastatic process. Results here highlight the potential of integrating these molecular markers with existing prognostic tools to enhance the accuracy of metastasis prediction in PCa. By identifying patients at risk for metastasis, clinicians can tailor treatment strategies more effectively, potentially improving survival outcomes and quality of life. This study underscores the importance of further research into the role of intratumoral biomarkers in PCa management. [ABSTRACT FROM AUTHOR]

Abstract Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer. Synopsis: PARP inhibitor (PARPi) resistance is a major treatment challenge that dramatically shortens patient survival. Using new mouse models of PARPi response and recurrence, we identified FLT1 as a potential biomarker and therapeutic target for reversing PARPi resistance in BRCA-mutant breast cancer. New mouse models were developed that recapitulate the PARPi response and recurrence observed in patients. A novel PARPi-adaptive resistance mechanism driven by the PGF-FLT1-AKT pathway was identified. FLT1 signaling protected the cells from PARPi-induced death by activating AKT pro-survival pathways and by dampening the cytotoxic immune response. Blocking FLT1 signaling, either genetically or pharmacologically using axitinib, re-sensitized PARPi-resistant tumors to PARPi treatment in mice. High FLT1 activation in tumor cells at pre-treatment significantly correlated with shorter progression-free survival on PARPi in patients with breast cancer. PARP inhibitor (PARPi) resistance is a major treatment challenge that dramatically shortens patient survival. Using new mouse models of PARPi response and recurrence, we identified FLT1 as a potential biomarker and therapeutic target for reversing PARPi resistance in BRCA-mutant breast cancer. [ABSTRACT FROM AUTHOR]

Background. Vascular endothelial growth factor A (VEGFA) is one of the most important factors for regulation of hematopoietic stem cells differentiation. It is involved in leukemogenesis and central nervous system (CNS) damage in acute leukemia. According to the literature, the VEGFA production by blast cells is increased, but the values of serum concentration and the associations with CNS involvement are contradictory.Aim. evaluate the VEGFA, VEGFR1, VEGFR2 concentration in serum and cerebrospinal fluid of patient with different types of acute leukemia in disease onset and during treatment.Materials and methods. The concentration of VEGFA in serum and cerebrospinal fluid was studied in 74 primary patients with acute leukemia. The comparison group consisted of 67 healthy donors. VEGFR1, VEGFR2 were studied in serum and cerebrospinal fluid in 34 patients at the onset of the disease. The comparison group consisted of 10 healthy donors. For the analysis, an enzyme immunoassay was used on a semi-automatic Personal Lab analyzer (Adaltis) and Affymetrix eBioscience human VEGF-A Platinum ELISA reagents.Results. Serum VEGFA concentration was statistically significantly lower in acute leukemia patients than that of donors (median 149.78 and 432.19 pg/ml respectively; p

Background: Prostate cancer (PCa) is the second most commonly diagnosed cancer in men worldwide, with metastasis, particularly to bone, being the primary cause of mortality. Currently, prognostic markers like PSA levels and Gleason classification are limited in predicting metastasis, emphasizing the need for novel clinical biomarkers. New molecules predicting tumor progression have been identified over time. Some, such as the immune checkpoint inhibitors (ICIs) PD-1/PD-L1, have become valid markers as theranostic tools essential for prognosis and drug target therapy. However, despite the success of ICIs as an anti-cancer therapy for solid tumors, their efficacy in treating bone metastases has mainly proven ineffective, suggesting intrinsic resistance to this therapy in the bone microenvironment. This study explores the potential of immunological intratumoral biomarkers, focusing on placental growth factor (PlGF), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), and Programmed Cell Death Protein 1 (PD-1), in predicting bone metastasis formation. Methods: we analyzed PCa samples from patients with and without metastasis by immunohistochemical analysis. Results: Results revealed that PlGF expression is significantly higher in primary tumors of patients that developed metastasis within five years from the histological diagnosis. Additionally, PlGF expression correlates with increased VEGFR1 and PD-1 levels, as well as the presence of intratumoral M2 macrophages. Conclusions: These findings suggest that PlGF contributes to an immunosuppressive environment, thus favoring tumor progression and metastatic process. Results here highlight the potential of integrating these molecular markers with existing prognostic tools to enhance the accuracy of metastasis prediction in PCa. By identifying patients at risk for metastasis, clinicians can tailor treatment strategies more effectively, potentially improving survival outcomes and quality of life. This study underscores the importance of further research into the role of intratumoral biomarkers in PCa management.

Passive transfer of antithyroid antibodies in mice leads to reproductive disorders. The purpose was to assess the placental tissue of experimental animals under the influence of the circulating thyroperoxidase antibodies. We performed an immunohistochemical examination of murine placentae after a passive transfer of thyroperoxidase antibodies. Placentae of mice that passively transferred IgG from healthy donors were used as control samples. For histological examination, 30 placental samples were selected from mice from the anti-TPO group and 40 placental samples were taken from mice from the IgG group. Immunostaining for VEGFR1, THBS 1, Laminin, CD31, CD34, FGF-β, CD56, CD14, TNF-α, kisspeptin, MCL 1, and Annexin V was performed. There is a significant decrease in the relative area of the expression of VEGFR1 (23.42 ± 0.85 vs. 33.44 ± 0.35, P < 0.01), thrombospondin 1 (31.29 ± 0.83 vs. 34.51 ± 0.75, P < 0.01), CD14 (25.80 ± 0.57 vs. 32.07 ± 0.36, P <.01), CD56 (30.08 ± 0.90 vs. 34.92 ± 0.15, P < 0.01), kisspeptin (25.94 ± 0.47 vs. 31.27 ± 0.57, P < 0.01), MCL 1 (29.24 ± 1.06 vs. 38.57 ± 0.79, P < 0.01) in the labyrinth zone of the placentae of mice from the anti-TPO group compared with control group. A significant increase in the relative expression of laminin and FGF-β was noted in the group of mice to which antibodies to thyroperoxidase were transferred, compared with the control group (36.73 ± 1.38 vs. 29.83 ± 0.94, P < 0.01 and 23.26 ± 0.61 vs. 16.38 ± 1.01, P < 0.01respectively). Our study exposed an imbalance of pro- and anti-angiogenic factors, decreased representation of placental macrophages and NK cells, abnormal trophoblast invasion processes, and insufficient expression of antiapoptotic factors in the placentae of mice in which anti-TPO antibodies were passively transferred. We performed an immunohistochemical examination of murine placentae after a passive transfer of thyroperoxidase antibodies. Our study exposed an imbalance of pro- and anti-angiogenic factors, decreased representation of placental macrophages and NK cells, abnormal trophoblast invasion processes, and insufficient expression of antiapoptotic factors in the placentae of mice in which anti-TPO antibodies were passively transferred. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) accounts for approximately 50% of end-stage renal diseases. Vascular endothelial growth factor A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in DN, but its role is unclear. The lack of pharmacological tools to modify renal concentrations further hinders the understanding of its role in DN. In this study, rats were evaluated after 3 weeks of streptozotocin-induced diabetes and two suramin treatments (10 mg/kg, ip). Vascular endothelial growth factor A expression was evaluated by western blot of glomeruli and immunofluorescence of the renal cortex. RT-PCR for receptors Vegfr1 mRNA and Vegfr2 mRNA quantitation was performed. The soluble adhesive molecules (sICAM-1, sVCAM-1) in blood were measured by ELISA and the vasoreactivity of interlobar arteries to acetylcholine was evaluated using wire myography. Suramin administration reduced the expression and intraglomerular localisation of VEGF-A. Increased VEGFR-2 expression in diabetes was reduced by suramin to non-diabetic levels. Diabetes reduced the sVCAM-1 concentrations. Suramin in diabetes restored acetylcholine relaxation properties to non-diabetic levels. In conclusion, suramin affects the renal VEGF-A/VEGF receptors axis and has a beneficial impact on endothelium-dependent relaxation of renal arteries. Thus, suramin may be used as a pharmacological agent to investigate the potential role of VEGF-A in the pathogenesis of renal vascular complications in short-term diabetes. [ABSTRACT FROM AUTHOR]

Abstract Background Inhibition of tumor angiogenesis through simultaneous targeting of vascular endothelial growth factor receptor (VEGFR)-1 and -2 is highly efficacious. An antagonist peptide of VEGFA/VEGFB, referred to as VGB3, can recognize and neutralize both VEGFR1 and VEGFR2 on the endothelial and tumoral cells, thereby inhibits angiogenesis and tumor growth. However, improved efficacy and extending injection intervals is required for its clinical translation. Given that gold nanoparticles (GNPs) can enhance the efficacy of biotherapeutics, we conjugated VGB3 to GNPs to enhance its efficacy and extends the intervals between treatments without adverse effects. Results GNP–VGB3 bound to VEGFR1 and VEGFR2 in human umbilical vein endothelial (HUVE) and 4T1 mammary carcinoma cells. GNP–VGB3 induced cell cycle arrest, ROS overproduction and apoptosis and inhibited proliferation and migration of endothelial and tumor cells more effectively than unconjugated VGB3 or GNP. In a murine 4T1 mammary carcinoma tumor model, GNP–VGB3 more strongly than VGB3 and GNP inhibited tumor growth and metastasis, and increased animal survival without causing weight loss. The superior antitumor effects were associated with durable targeting of VEGFR1 and VEGFR2, thereby inhibiting signaling pathways of proliferation, migration, differentiation, epithelial-to-mesenchymal transition, and survival in tumor tissues. MicroCT imaging and inductively coupled plasma mass spectrometry showed that GNP–VGB3 specifically target tumors and exhibit greater accumulation within tumors than the free GNPs. Conclusion Conjugation to GNPs not only improved the efficacy of VGB3 peptide but also extended the intervals between treatments without adverse effects. These results suggest that GNP–VGB3 is a promising candidate for clinical translation. Graphical Abstract

Acute lung injury (ALI) is characterized by hyperinflammation followed by vascular leakage and respiratory failure. Vascular endothelial growth factor (VEGF)-A is critical for capillary permeability; however, the role of VEGF receptor 1 (VEGFR1) signaling in ALI progression remains unclear. Here, we show that deletion of VEGFR1 tyrosine kinase (TK) signaling in mice exacerbates lipopolysaccharide (LPS)-induced ALI as evidenced by excessive pro-inflammatory cytokine production and interleukin(IL)-1β-producing neutrophil recruitment to inflamed lung tissues. ALI development involves reduced alveolar macrophage (AM) levels and recruitment of monocyte-derived macrophages (MDMs) in a VEGFR1 TK-dependent manner. VEGFR1 TK signaling reduced pro-inflammatory cytokine levels in cultured AMs. VEGFR1 TK-expressing MDMs displayed an anti-inflammatory macrophage phenotype. Additionally, the transplantation of VEGFR1 TK-expressing bone marrow (BM)-derived macrophages into VEGFR1 TK-deficient mice reduced lung inflammation. Treatment with placental growth factor (PlGF), an agonist for VEGFR1, protected the lung against LPS-induced ALI associated with increased MDMs. These results suggest that VEGFR1 TK signaling prevents LPS-induced ALI by suppressing the pro-inflammatory activity of AMs and enhancing the anti-inflammatory function of MDMs. • Abrogation of VEGFR1 TK signaling exacerbates acute lung injury. • VEGFR1 TK-deficient AMs enhance cytokine production upon LPS stimulation. • VEGFR1 TK-expressing MDMs mitigate acute lung injury. • Activation of VEGFR1 protects the lung against acute lung injury. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1 and R2) play important roles in the progression of malignant melanoma through tumor angiogenesis. However, it is not clear whether the VEGF-A/VEGFR1 signaling pathway is involved in the proliferation and migration of melanoma cells. Thus, the effect of VEGF-A on cell migration was investigated in human melanoma cell lines. Of several splicing variants of VEGF-A, VEGF165 is the most abundant and responsible for VEGF-A biological potency. VEGF165 facilitated the migration of melanoma cells in both a chemotactic and chemokinetic manner, but cell proliferation was not affected by VEGF165. VEGF165 also induced the phosphorylation of Akt. In addition, VEGF165-induced cell migration was inhibited significantly by VEGFR1/2 or a VEGFR1-neutralizing antibody. Furthermore, the downregulation of VEGFR1 via the transfection of VEGFR1-targeting antisense oligonucleotides suppressed VEGF165-induced cell migration. Moreover, wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI3K) in the PI3K/Akt pathway, suppressed VEGF165-induced Akt phosphorylation and VEGF165-induced cell migration. These findings suggest that the motility of melanoma cells is regulated by signals mediated through the PI3K/Akt kinase pathway with the activation of VEGFR1 tyrosine kinase by VEGF165. Thus, the downregulation of signaling via VEGF-A/VEGFR1 might be an effective therapeutic approach that could prevent the progression of malignant melanoma. [ABSTRACT FROM AUTHOR]

Neurons in the penumbra (the area surrounding ischemic tissue that consists of still viable tissue but with reduced blood flow and oxygen transport) may be rescued following stroke if adequate perfusion is restored in time. It has been speculated that post-stroke angiogenesis in the penumbra can reduce damage caused by ischemia. However, the mechanism for neovasculature formation in the brain remains unclear and vascular-targeted therapies for brain ischemia remain suboptimal. Here, we show that VEGFR1 was highly upregulated in pericytes after stroke. Knockdown of VEGFR1 in pericytes led to increased infarct area and compromised post-ischemia vessel formation. Furthermore, in vitro studies confirmed a critical role for pericyte-derived VEGFR1 in both endothelial tube formation and pericyte migration. Interestingly, our results show that pericyte-derived VEGFR1 has opposite effects on Akt activity in endothelial cells and pericytes. Collectively, these results indicate that pericyte-specific expression of VEGFR1 modulates ischemia-induced vessel formation and vascular integrity in the brain. [ABSTRACT FROM AUTHOR]

Angiogenesis is a critical process during human skin wound healing. However, hypoxia might lead to the dysfunction of the cellular interplay of endothelial cells and subcutaneous fibroblasts, resulting in the deregulation of angiogenesis. HIF1A is a key regulatory of the recovery of intracellular homeostasis under hypoxia. In the present study, the detailed role and mechanism of HIF1A in the angiogenesis under hypoxia were investigated. Via bioinformatic analyses on microarray profiles (GSE1041 and GSE17944), solube fms-related tyrosine kinase 1 (sFLT1, also known as sVEGFR1) and miR-210/miR-424 might be involved in HIF1A function on the angiogenesis under hypoxia in human umbilical vascular endothelium cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs). In the present study, we identified sFLT1 as a downregulated gene in response to hypoxia and HIF1A overexpression in HUVECs and HDMECs. sFLT1 overexpression inhibited the capacity of migration and angiogenesis and significantly reversed the inducible effects of HIF1A on the migration and angiogenesis in both cell lines. miR-210 and miR-424 were upregulated by hypoxia and targeted sFLT1 3′-UTR to negatively modulate its expression. HIF1A modulated sFLT1 expression, VEGF signaling, and the migration and angiogenesis in HUVECs and HDMECs via miR-210/miR-424. Regarding the molecular mechanism, HIF1A bound the promoter region of miR-210 and miR-424 to activate their transcription, while miR-210/miR-424 bound sFLT1 3′-UTR to suppress its expression. In summary, HIF1A/miR-210/miR-424/sFLT1 axis modulates the angiogenesis in HUVECs and HDMECs upon hypoxic condition via VEGF signaling. [ABSTRACT FROM AUTHOR]

It has been demonstrated that vascular endothelial growth factor B (VEGFB) and vascular endothelial growth factor receptor 1 (VEGFR1) play a vital role in regulating vascular biological function. However, the role of VEGFB and VEGFR1 in regulating fat deposition and skeletal muscle growth remains unclear. Therefore, this study was conducted to investigate the effects of VEGFB and VEGFR1 on fat deposition and skeletal muscle growth in mice. Our results showed that knockdown of VEGFB decreased body weight and iWAT index, stimulated the browning of mice iWAT with increased expression of UCP1, decreased the diameters of adipocytes, and elevated energy expenditure. In contrast, knockdown of VEGFB increased gastrocnemius (GAS) muscle index with increased proliferation of GAS muscle by expression of PCNA and Cyclin D1. Meanwhile, knockdown of endothelial VEGFR1 induced the browning of iWAT with increased expression of UCP1 and decreased diameters of adipocytes. By contrast, knockdown of endothelial VEGFR1 inhibited GAS muscle differentiation with decreased expression of MyoD. In conclusion, these results suggested that the loss of VEGFB/VEGFR1 signaling is associated with enhanced browning of inguinal white adipose tissue and skeletal muscle development. These results provided new insights into the regulation of skeletal muscle growth and regeneration, as well as fat deposition, suggesting the potential application of VEGFB/VEGFR1 as an intervention for the restriction of muscle diseases and obesity and related metabolic disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Squamous cell carcinoma (SCC) is a malignant skin cancer that affects domestic animal species and humans with similar characteristics. Our research seeks to understand the mechanisms by which SCC progression depends on the development of a new blood supply (angiogenesis) in the host. Here, we queried our archive of cat SCC tumor samples to measure expression of genes coding for angiogenic signaling proteins that can exist in closely related forms with distinct biological properties. We observed that, when compared to normal skin, SCC tissues contained a greater abundance of gene transcripts encoding a form of the growth factor PLGF, predicted to have an altered distribution in the body. Similarly, altered patterns of expression were observed for forms of the PLGF receptor Flt-1, which can modulate angiogenesis. Future studies will test the relationship between these gene expression changes and the severity of SCC in order to establish them as predictive biomarkers of SCC progression in individual patients. Cutaneous squamous cell carcinoma (CSCC) is a common malignant skin cancer with a significant impact on health, and it is important to determine the degree of reliance of CSCC on angiogenesis for growth and metastasis. Major regulators of angiogenesis are the vascular endothelial growth factor (VEGF) family and their associated receptors. Alternative pre-mRNA splicing produces multiple isoforms of VEGF-A and PLGF with distinct biological properties. Several studies highlight the function of VEGF-A in CSCC, but there are no studies of the different isoforms of VEGF-A and PLGF for this neoplasm. We characterized the expression of three isoforms of VEGF-A, two isoforms of PLGF, and their receptors in cat CSCC biopsies compared to normal haired skin (NHS). Although our results revealed no significant changes in transcript levels of panVEGF-A or their isoforms, the mRNA levels of PLGF I and the receptors Flt-1 and KDR were downregulated in CSCC compared to NHS. Differences were observed in ligand:receptor mRNA expression ratio, with the expression of VEGF-A relative to its receptor KDR higher in CSCC, which is consistent with our hypothesis and prior human SCC studies. Immunolocalization in tissue showed increased expression of all measured factors and receptors in tumor cells compared to NHS and surrounding vasculature. We conclude that the factors measured may play a pivotal role in CSCC growth, although further studies are needed to clarify the role of angiogenic factors in feline CSCC. [ABSTRACT FROM AUTHOR]

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases. [ABSTRACT FROM AUTHOR]

There is ample evidence to suggest that vascular endothelial growth factor (VEGF) is a potent mitogen factor in vasculogenesis and angiogenesis and that blockade of VEGF-mediated signals can also prevent tumor growth via enforcing cell apoptosis. In the current study, we assessed the suppressing effect of VGB4, a VEGF antagonist peptide with the binding ability to both VEGF receptor1 and VEGF receptor2, on VEGF-induced proliferation and migration of the human lung adenocarcinoma cell line A549 and the human colon adenocarcinoma cell line HT29 using MTT assay, colony formation assay, and Scratch-wound assay. To evaluate the apoptotic inductive effect of VGB4 on A549 and HT29 cells, apoptosis analysis was carried out by flow cytometry and TUNEL assay. Likewise, p53 and PTEN expression level was examined by immunofluorescence microscopy. In addition, the level of proteins involved in VEGF signaling pathways related to apoptosis was investigated using western blot analysis. Our results indicated that VGB4 markedly inhibited VEGF-induced proliferation and migration, and induced apoptosis of A549 and HT29 cells dose dependently. Encouragingly, significant downregulation of B-cell lymphoma 2 (Bcl2), X-linked inhibitor of apoptosis, Procaspase9, and procaspase3, as well as upregulation of PTEN and P53 tumor suppressors, BCL2 associated X, Cytochrome c, cleaved caspase9, and cleaved caspase3 in VGB4-treated A549 and HT29 cells, further confirmed the profound inductive influence of VGB4 on apoptotic pathways. These findings along with the results from our previous studies show that VGB4 may be considerable for cancer therapy. [ABSTRACT FROM AUTHOR]

• ZAM administration had no effect on the bodyweight of cervical cancer rats. • Antioxidants found in andaliman can lower levels of MDA and serum NGAL, thereby increasing SOD activity. • ZAM treatment can suppress the production of IL1β and TGFβ1 which promotes cancer cell growth in rats. • ZAM administration can increase IL-10 expression in cervical cancer rats, thereby suppressing the growth of cervical cancer. • ZAM decrease VEGFR1 serum expression and improve histology in cervical cancer rats. Cervical cancer is the second most lethal cancer in Indonesia, behind breast cancer. One of the reasons cancer cells are difficult to treat is that the immune system is sometimes unable to recognise them as foreign. Cytokinin therapy is carried out so that the immune system can strengthen its response to cancer cells, with the aim of slowing or stopping the development of malignant cells. Zanthoxylum acanthopodium DC, also known as andaliman, is an Indonesian herb and a member of the Rutaceae family. It is rich in antioxidants and has anti-inflammatory and anti-cancer properties. The current study aimed to investigate the histological changes and changes in the expression of cytokines, such as IL-10, IL1β, VEGFR1, and TGFβ1, associated with andaliman treatment. Sample tissues and serums extracted from cervical cancer rat models were used. Rats were divided into five groups: a control group (C−), cancer model group (C+), cancer with a dose of Z. acanthopodium methanolic extract (ZAM) 100 mg/body weight (BW) ZAM (ZAM100), cancer with a dose of ZAM 200 mg/BW ZAM (ZAM200), and cancer with a dose of ZAM 400 mg/BW ZAM (ZAM400). Treatment lasted for 1 month. Blood samples were prepared for ELISA analysis, and cervical tissue was stained for immunohistochemistry using antibodies against IL-10, IL-1β, VEGFR1, and TGFβ1. Administration of ZAM had no significant effect on rat body weight and cervical organs (p > 0.05). However, it impacted haematological parameters in rats with cervical cancer (p < 0.05). Elevated malondialdehyde levels may be linked to superoxide dismutase deficiency in tumour tissue. ZAM significantly decreased the expression of IL1β, TGFβ1, and VEGFR1 (p < 0.01), while it increased the expression of IL-10. Therefore, ZAM may be a potential target for molecular cytokine therapy for cervical cancer. [ABSTRACT FROM AUTHOR]

Lymphangiogenesis is related to the growth of endometriosis. Here, we examined whether vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) signaling plays a role in lymphangiogenesis during endometriosis. Endometrial fragments from wild-type (WT) mice transplanted into the peritoneal wall of host WT mice (WT→WT) developed well and displayed enhanced lymphangiogenesis associated with increases in mRNA levels of VEGF-C and VEGF-D. Compared with WT mice, the implant size and lymphangiogenesis were reduced, when endometrial fragments from mice lacking the VEGFR1 tyrosine kinase (TK) domain (TK-/-) were transplanted into host TK-/- mice (TK-/-→TK-/-). Treatment of WT→WT mice with the VEGFR3 kinase inhibitor suppressed the size of implants and lymphangiogenesis. Immunofluorescence analyses demonstrated that VEGF-C and VEGF-D were expressed in both CD11b+ and S100A4+ cells. TK-/-→TK-/- mice had lower numbers of CD11b+ and S100A4+ cells than WT→WT mice. When isolated bone marrow (BM)-derived macrophages or culture murine fibroblasts were stimulated with placental growth factor (PlGF), a specific agonist of VEGFR1, the levels of VEGF-C and VEGF-D were increased in a VEGFR1-dependent manner. These results suggest that VEGFR1 signaling in macrophages and fibroblasts contributes to the growth of endometrial implants and lymphangiogenesis.

Background: Evaluation and search for new molecular markers of renal cell cancer, first of all, associated with angiogenic and invasive activity, continue to be highly relevant. In our previous publications, we have assessed the potential diagnostic value of matrix metalloproteinases (MMP) 2, 7, 8, and 9, their tissue inhibitor type 1 (TIMP1) and components of the VEGF signaling system in renal cell cancer. Aim: To assess the role of serum VEGF, VEGFR1, VEGFR2, MMP2, 7, 8, 9, and TIMP1 levels in renal cell patients as diagnostic and prognostic markers of overall survival. Materials and methods: 99 renal cell cancer patients (94 primary and 5 at progression) were recruited into the study. The control group included 97 healthy control blood donors. Ninety three (93) primary patients with renal cell cancer were followed for 1 to 45 (median, 26) months for assessment of their overall survival. Serum concentrations of the study proteins were measured by direct immunoenzyme analysis (Quantikine® ELISA kits, R&D Systems, США). Results: Serum VEGF, VEGFR1, VEGFR2, MMP7, MMP8, and TIMP1 levels in renal cell cancer patients are significantly higher than those in the control group. The diagnostic characteristics of the markers are considerably different, the most reliable marker with 84% sensitivity at 87.5% specificity being MMP7. VEGFR1, MMP7, MMP8, and TIMP1 were positively associated with disease stage and TNM indices. MMP7 and TIMP1 levels also increased with a higher tumor grade. MMP7 was found to be a significant unfavorable prognostic factor for overall survival: the 3-years survival in those with low (< 6.3 ng/ml) marker level amounted to 93%, whereas with high, 51% (p < 0.001). MMP7 prognostic value remained significant also in stage I renal cancer: after 3-years' follow-up, all patients with low MMP7 were alive, while survival of those with high marker levels was 72% (p=0.02). Increased serum MMP8 level (> 51 ng/ml) also had an unfavorable prognostic value in the whole renal cell cancer patient group, with 3-years' survival being 78 and 58% for low and high levels, respectively (p < 0.01). The components of VEGF signaling system, MMP2, MMP9, and TIMP1 had no significant prognostic values. Conclusion: MMP7 should be viewed as the most promising diagnostic and prognostic renal cell cancer marker. VEGF and its soluble receptors could be useful for monitoring of patients receiving anti-angiogenic treatments and prediction of their sensitivity to these agents.

We previously demonstrated that a deficiency of natural antibodies against CD25, Mucin 1 (MUC1), and vascular endothelial growth factor receptor 1 (VEGFR1) could contribute to high risk of non‐small cell lung cancer (NSCLC). This study was designed to investigate whether natural IgG antibodies against POU domain class 5 transcription factor 1 (POU5F1), tumor necrosis factor‐α (TNF‐α), and the combination of CD25, VEGFR1, and MUC1 could play an anti‐tumorigenic role against developing NSCLC. An ELISA was developed in‐house to examine plasma IgG against peptide antigens derived from POU5F1, TNF‐α, and a combination of peptide antigens derived from CD25, MUC1, and VEGFR1 in 211 patients with NSCLC and 200 healthy controls. Mann–Whitney U test demonstrated that plasma IgG levels for the combination of peptide antigens derived from CD25, MUC1, and VEGFR1 were significantly lower in NSCLC patients than control subjects (Z = −12.978, P

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.

Background: Inhibition of tumor angiogenesis through simultaneous targeting of vascular endothelial growth factor receptor (VEGFR)-1 and -2 is highly efficacious. An antagonist peptide of VEGFA/VEGFB, referred to as VGB3, can recognize and neutralize both VEGFR1 and VEGFR2 on the endothelial and tumoral cells, thereby inhibits angiogenesis and tumor growth. However, improved efficacy and extending injection intervals is required for its clinical translation. Given that gold nanoparticles (GNPs) can enhance the efficacy of biotherapeutics, we conjugated VGB3 to GNPs to enhance its efficacy and extends the intervals between treatments without adverse effects. Results: GNP–VGB3 bound to VEGFR1 and VEGFR2 in human umbilical vein endothelial (HUVE) and 4T1 mammary carcinoma cells. GNP–VGB3 induced cell cycle arrest, ROS overproduction and apoptosis and inhibited proliferation and migration of endothelial and tumor cells more effectively than unconjugated VGB3 or GNP. In a murine 4T1 mammary carcinoma tumor model, GNP–VGB3 more strongly than VGB3 and GNP inhibited tumor growth and metastasis, and increased animal survival without causing weight loss. The superior antitumor effects were associated with durable targeting of VEGFR1 and VEGFR2, thereby inhibiting signaling pathways of proliferation, migration, differentiation, epithelial-to-mesenchymal transition, and survival in tumor tissues. MicroCT imaging and inductively coupled plasma mass spectrometry showed that GNP–VGB3 specifically target tumors and exhibit greater accumulation within tumors than the free GNPs. Conclusion: Conjugation to GNPs not only improved the efficacy of VGB3 peptide but also extended the intervals between treatments without adverse effects. These results suggest that GNP–VGB3 is a promising candidate for clinical translation. [ABSTRACT FROM AUTHOR]

Estudiar la posible relación entre la expresión inmunohistoquímica del receptor 1 del factor de crecimiento endotelial vascular (VEGFR1) y el valor máximo de captación estandarizada (SUV máx) de la PET 18F-FDG en pacientes con cáncer de pulmón de células no pequeñas. El estudio incluyó 39 pacientes con NSCLC (24 carcinomas de células escamosas y 15 adenocarcinomas). Según el estadio clínico, los pacientes se distribuyeron de la siguiente manera: 8 en estadio I, 7 en estadio II, 15 en estadio III y 9 en estadio IV. Se estudió la expresión inmunohistoquímica del VEGFR1 mediante la técnica de la matriz tisular utilizando el dispositivo de arreglo de tejidos (Beecher Instruments, Sun Prairie, WI), utilizando el anticuerpo policlonal contra el VEGFR1 (Santa Cruz Biotechnology, California, EE. UU.). Se observó una expresión inmunohistoquímica positiva del VEGFR1 en 23 casos (59%). El número de tumores positivos no se relacionó con el estadio clínico pero hubo una asociación estadísticamente significativa diferente (p: 0,0009) entre la positividad de VEGFR1 y el tipo histológico, correspondiendo los mayores porcentajes de resultados positivos a los adenocarcinomas (93,3%) frente a los carcinomas escamocelulares (37,5%). Asimismo, los valores SUV máx fueron mayores (p: 0,039) en los carcinomas VEGFR1 negativos que en los tumores VEGFR1 positivos (r: 4-32,1; 16,4 + /-6,4 [mediana 16,1] vs. r: 3-47; 14,5 + /-8,6 [12,8]). Nuestros resultados nos llevaron a considerar que en el CPCNP, la expresión inmunohistoquímica negativa de VEGFR1 se asocia significativamente con el subtipo de carcinomas de células escamosas y con valores SUV máx más altos en 18F-FDG-PET. To study the possible relation between immunohistochemical expression of vascular endothelial growth factor receptor 1 (VEGFR1) and the maximum standardised uptake value (maxSUV) of 18F-FDG PET in patients with non small cell lung cancer. The study included 39 patients with NSCLC (24 squamous cell carcinomas and 15 adenocarcinomas). According to the clinical stage, the patients were distributed as follows: 8 stage I, 7 stage II, 15 stage III and 9 stage IV. Immunohistochemical expression of VEGFR1 was studied through the technique of tissue-matrix using tissue arrayer device (Beecher Instruments, Sun Prairie, WI), using the polyclonal antibody against VEGFR1 (Santa Cruz Biotechnology, California, USA). Positive VEGFR1 immunohistochemical expression was noted in 23 cases (59%). The number of positive tumours was not related with clinical stage but there was a different statistically significant association (p:.0009) between VEGFR1 positivity and histological type, corresponding the greater percentages of positive results to adenocarcinomas (93.3%) versus in squamous cell carcinomas (37.5%). Likewise, maxSUV values were higher (p:.039) in negative VEGFR1 carcinomas than in positive VEGFR1 tumors (r: 4-32.1; 16.4 + /-6.4 [median 16.1] vs. r: 3-47; 14.5 + /-8.6 [12.8]). Our results led us to consider that in NSCLC, the negative VEGFR1 immunohistochemical expression is associated significantly with squamous cell carcinomas subtype and with higher maxSUV values in 18F-FDG-PET. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor (VEGF) is one of the primary angiogenesis regulators in solid cancers. Brain solid tumors are life-threatening diseases in which angiogenesis is an important phase of tumor development and progression. In the present study, VEGF-A and VEGF receptor (VEGF-R1) gene expression was evaluated in CNS brain tumors. Methods: VEGF-A and VEGF-R1 expression was quantified using real-time PCR on fresh biopsies of 38 supratentorial brain tumors compared to 30 non-tumoral tissues. Then, the correlations were investigated with clinic-pathological and demographic factors of the patients. Results: PCR product sequencing confirmed the validity of qRT-PCR. Although VEGF-A and VEGF-R1 expression showed increasing trends with the progression of cell proliferation in different stages of astrocytoma, VEGF-R1 did not meet the 95% confidence interval in other brain tumors. An increasing trend in VEGF-A expression and a declining trend in VEGF-R1 expression from Stage I to II were observed in meningioma. VEGF-A and VEGF-R1 expression had no significant correlation with age and gender. Although peritumoral brain edema (PTBE) in astrocytoma was significantly associated with tumor stages, VEGF-A and VEGF-R1 were not correlated with PTBE in meningioma and metastasis. Conclusions: VEGF-A is a valuable factor for the prognosis of PTBE and malignancy in astrocytoma and is helpful in monitoring treatment approaches. [ABSTRACT FROM AUTHOR]

Purpose: Triple-negative breast cancer (TNBC) is specified by high vascularity and repetitious metastasis. Although several studies have indicated that angiogenesis has an important role in invasive breast cancer, a suitable model of TNBC that can show the exact onset of angiogenesis factors still needs to be developed. The purpose of this study is to determine the expression level of angiogenesis factors in different clinical stages of the 4T1 tumor as TNBC mouse model. Methods: Twenty mice were injected by the 4T1 cell line, and four mice selected as healthy controls. Following by tumor induction, the mice were randomly put into four groups, each contains four mice. Once the tumor volume reached to the early stage (500 mm3 ), they were removed by surgery. Then, the expression levels of Hif1α, VEGFR1, and VEGFR2 genes, as well as tumor markers of VEGF, bFGF and CD31, were evaluated by qPCR and immunohistochemistry (IHC) respectively. The statistical analysis was done by SPSS version 16. Results: TNBC tumors were confirmed and multi-foci metastasis in the lung were seen. The mRNA and protein expression levels of the angiogenesis factors increased in the early stage and as the tumor grew, their expression level enhanced dramatically. Conclusion: The 4T1 syngeneic mouse tumor may serve as an appropriate TNBC model for further investigation of the angiogenesis and therapies. Moreover, angiogenesis factors are induced before the advanced stage, and anti-angiogenesis therapy is necessary to be considered at the first line of treatment in TBNC.

Objective: Vascular endothelial growth factor A (VEGFA) and its receptors are essential proteins for the angiogenic activity of dental pulp. Angiogenesis fundamentally provides oxygen and nutrients to cells for root formation and defence mechanisms. The angiogenic potential of dental pulp should be understood and considered for the conservative and regenerative endodontics. The purpose of this research was to measure the VEGFA expression and its receptors such as vascular endothelial growth factor receptors 1, -2 (VEGFR1, VEGFR2) and Neuropilin 1 (NRP1) in human dental pulp from molars with immature and mature apexes. Methods: VEGFA system mRNAs expressions were assessed in dental pulp obtained from freshly extracted human third molars divided into immature (n=8) and mature (n=8) apexes. RNAs were extracted from the samples. Each sample's cDNA was synthetized and the target genes VEGFA, VEGFR1, VEGFR2, NRP1 expression profiles obtained by RT2-PCR. Analysis was based on the Student's t-test comparing the replicate 2-?Ct values for each gene. P values of <0.05 were considered significant. Results: In teeth with mature apexes, VEGFA (P=0.0002), NRP1 (P=0.0001), VEGFR1 (P=0.0057) and VEGFR2 (P=0.018259) significantly increased statistically with respect to the immature apexes group. Conclusion: Within the limitation of the present investigation, it can be concluded that the angiogenic process seems to be a physiological process in the dental pulp due to the studied angiogenic growth factor are expressed in both immature and mature dental pulps. VEGFA and its receptors are expressed significantly higher in mature apex teeth than immature apex teeth. [ABSTRACT FROM AUTHOR]

Cutaneous squamous cell carcinoma (CSCC) is a common malignant skin cancer with a significant impact on health, and it is important to determine the degree of reliance of CSCC on angiogenesis for growth and metastasis. Major regulators of angiogenesis are the vascular endothelial growth factor (VEGF) family and their associated receptors. Alternative pre-mRNA splicing produces multiple isoforms of VEGF-A and PLGF with distinct biological properties. Several studies highlight the function of VEGF-A in CSCC, but there are no studies of the different isoforms of VEGF-A and PLGF for this neoplasm. We characterized the expression of three isoforms of VEGF-A, two isoforms of PLGF, and their receptors in cat CSCC biopsies compared to normal haired skin (NHS). Although our results revealed no significant changes in transcript levels of panVEGF-A or their isoforms, the mRNA levels of PLGF I and the receptors Flt-1 and KDR were downregulated in CSCC compared to NHS. Differences were observed in ligand:receptor mRNA expression ratio, with the expression of VEGF-A relative to its receptor KDR higher in CSCC, which is consistent with our hypothesis and prior human SCC studies. Immunolocalization in tissue showed increased expression of all measured factors and receptors in tumor cells compared to NHS and surrounding vasculature. We conclude that the factors measured may play a pivotal role in CSCC growth, although further studies are needed to clarify the role of angiogenic factors in feline CSCC.

Introduction: Endometriosis is a chronic inflammatory disease that affects ∼10 % of women. A significant fraction of patients experience limited or no efficacy with current therapies. Tissue adjacent to endometriosis lesions often exhibits increased neurite and vascular density, suggesting that disease pathology involves neurotrophic activity and angiogenesis., Objectives: We aim to evaluate the potential for key tyrosine-kinase-receptor-coupled neurotrophic molecules to contribute to endometriosis-associated pain in mice., Methods: Peritoneal fluid was collected from endometriosis patients undergoing surgery and the levels of NGF and VEGFR1 regulators (VEGFA, VEGFB, PLGF, and sVEGFR1) were quantified by ELISA. VEGFR1 regulator concentrations were used to calculate VEGFR1 occupancy. We used genetic depletion, neutralizing antibodies, and pharmacological approaches to specifically block neurotrophic ligands (NGF or BDNF) or receptors (VEGFR1, TRKs) in a murine model of endometriosis-associated pain. Endometriosis-associated pain was measured using von Frey filaments, quantification of spontaneous abdominal pain-related behavior, and thermal discomfort. Disease parameters were evaluated by lesion size and prevalence. To evaluate potential toxicity, we measured the effect of entrectinib dose and schedule on body weight, liver and kidney function, and bone structure (via micro-CT)., Results: We found that entrectinib (pan-Trk inhibitor) or anti-NGF treatments reduced evoked pain, spontaneous pain, and thermal discomfort. In contrast, even though calculated receptor occupancy revealed that VEGFR1 agonist levels are sufficient to support signaling, blocking VEGFR1 via antibody or tamoxifen-induced knockout did not reduce pain or lesion size in mice. Targeting BDNF-TrkB with an anti-BDNF antibody also proved ineffective. Notably, changing dosing schedule to once weekly eliminated entrectinib-induced bone-loss without decreasing efficacy against pain., Conclusions: This suggests NGF-TrkA signaling, but not BDNF-TrkB or VEGF-VEGFR1, mediates endometriosis-associated pain. Moreover, entrectinib blocks endometriosis-associated pain and reduces lesion sizes. Our results also indicated that entrectinib-like molecules are promising candidates for endometriosis treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

As a vital male accessory reproductive gonad, the prostate requires vascular endothelial growth factors for promoting its growth and development. In this study, we investigated the localizations and expressions of vascular endothelial growth factor (VEGF) and its receptors including VEGF-receptor1 (VEFGR1) and VEGF-receptor2 (VEGFR2) in the prostate of the wild ground squirrels during the breeding and the non-breeding seasons. The values of total prostate weight and volume in the breeding season were higher than those in the non-breeding season. Histological observations showed that the exocrine lumens of the prostate expanded in the breeding season and contracted in the non-breeding season. The mRNA expression levels of VEGF and VEGFR2 in the prostate were higher in the breeding season than those in the non-breeding season, but the mRNA expression level of VEGFR1 had no significant change between the breeding and non-breeding seasons. Immunohistochemical results revealed that VEGF, VEGFR1 and VEGFR2 were presented in epithelial and stromal cells during the breeding and non-breeding seasons. In addition, the microvessels of the prostate were widely distributed and the number of microvessels increased obviously in the breeding season, while decreased sharply in the non-breeding season. These results suggested that expression levels of VEGF and VEGFR2 might be correlated with seasonal changes in morphology and functions of the prostate, and VEGF might serve as pivotal regulators to affect seasonal changes in the prostate functions of the wild male ground squirrels via an autocrine/paracrine pathway.

Abstract Objective During degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration. Results Expression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of the classic vascularization pathway is stable across degeneration stages and we assume that soluble vascular endothelial growth factor receptor 1 does not contribute to prevent disc degeneration. However, we observed increased expression levels of genes involved in alternative vascularization signalling pathways in severely degenerated discs, suggesting that abnormal vascularization is part of the pathological progression of disc degeneration.

• VEGF165b treatment augments the cytolytic ability of NK92 against tumor cells in vitro. • VEGF165b treatment upregulates perforin and granzyme B. • 3.VEGF165b activity is mediated by the VEGFR1-PLC pathway. Pro-angiogenic Vascular endothelial growth factors (VEGFs) exert immunosuppressive functions on some immune cells by interacting with VEGF receptors. Blocking the VEGF/VEGFR pathway could reverse the tumor immunosuppressive microenvironment to some degree. We recently demonstrated that the anti-angiogenic VEGF isoform VEGF165b, similar to other anti-angiogenic agents, inhibit the accumulation immunosuppressive cells such as Tregs and MDSCs. However, whether VEGF165b affects the functions of immune effector cells remain unclear. Here, NK92 cell line was utilized as an immune effector cell model. Our results verified that NK92 cells endogenously express VEGF165 and VEGFR1. Further investigation showed that NK92 treatment with VEGF165b augments its killing ability against human K562 leukemia cells by upregulating perforin and granzyme B through the VEGFR1-PLC pathway, whereas VEGF165b had no impact on the proliferation of NK92 cells in vitro. The results of this study improve our understanding of the immunomodulatory function of VEGF165b, which may help in enhancing the efficacy of NK92-based cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: Malignant neoplasm of the endometrium is the most common malignant neoplasm of the female reproductive system. Toll Like Receptors (TLR) play a significant role in innate and late-immunity against infections or damaged tissues. TLRs are also involved in the development of tumors in their natural microenvironment. TLRs play an important role in angiogenesis, necessary for survival and growth of the tumor. Hypoxia playing a critical role in angiogenesis, carcinogenesis, tumor progression and distant metastasis is primarily mediated through hypoxia inducible factors (HIFs). Vascular endothelial growth factor family proteins (VEGF) are also strongly involved in tumor angiogenesis and their action is strongly associated with TLR receptors. Objectives: The aim of the study was to correlate the expression of selected TLRs and VEGFR's as well as HIF1α with clinicopathological data of endometrial cancer patients. Material and methods: 123 neoplastic endometrial samples were included in the study. 51 samples of healthy endometrium served as control. The expression of TLR1, TLR2, TLR3, TLR4, VEGFR1 and VEGFR2, VEGF-A and HIF1α was examined after RNA isolation at the mRNA level by Real Time-PCR. Results: We have noted a significant correlation between the expression of selected TLR and VEGFR's and clinical stage as well as pathological grading of endometrial cancer. Conclusions: Received correlations confirm a significant contribution of some TLR expression and the receptor for VEGF in the pathogenesis of epithelial endometrial cancer. [ABSTRACT FROM AUTHOR]

We previously demonstrated that a deficiency of natural antibodies against CD25, Mucin 1 (MUC1), and vascular endothelial growth factor receptor 1 (VEGFR1) could contribute to high risk of non‐small cell lung cancer (NSCLC). This study was designed to investigate whether natural IgG antibodies against POU domain class 5 transcription factor 1 (POU5F1), tumor necrosis factor‐α (TNF‐α), and the combination of CD25, VEGFR1, and MUC1 could play an anti‐tumorigenic role against developing NSCLC. An ELISA was developed in‐house to examine plasma IgG against peptide antigens derived from POU5F1, TNF‐α, and a combination of peptide antigens derived from CD25, MUC1, and VEGFR1 in 211 patients with NSCLC and 200 healthy controls. Mann–Whitney U test demonstrated that plasma IgG levels for the combination of peptide antigens derived from CD25, MUC1, and VEGFR1 were significantly lower in NSCLC patients than control subjects (Z = −12.978, P < 0.001) although plasma levels of IgG antibodies for POU5F1 and TNFα were not significantly changed. The in‐house ELISA made with the CD25‐MUC1‐VEGFR1 combination had a sensitivity of 49.6% against a specificity of 95% to detect early‐stage NSCLC. In conclusion, natural antibodies against the combination of CD25, VEGFR1, and MUC1 may be an effective biomarker for early diagnosis of NSCLC. [ABSTRACT FROM AUTHOR]

Background: γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. Methods: For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. Result: These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. Conclusion: Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics. [ABSTRACT FROM AUTHOR]

Purpose: Triple-negative breast cancer (TNBC) is specified by high vascularity and repetitious metastasis. Although several studies have indicated that angiogenesis has an important role in invasive breast cancer, a suitable model of TNBC that can show the exact onset of angiogenesis factors still needs to be developed. The purpose of this study is to determine the expression level of angiogenesis factors in different clinical stages of the 4T1 tumor as TNBC mouse model. Methods: Twenty mice were injected by the 4T1 cell line, and four mice selected as healthy controls. Following by tumor induction, the mice were randomly put into four groups, each contains four mice. Once the tumor volume reached to the early stage (<100 mm3), intermediate stage (100-300 mm3), advanced stage (300-500 mm3), and end stage (>500 mm3), they were removed by surgery. Then, the expression levels of Hif1a, VEGFR1, and VEGFR2 genes, as well as tumor markers of VEGF, bFGF and CD31, were evaluated by qPCR and immunohistochemistry (IHC) respectively. The statistical analysis was done by SPSS version 16. Results: TNBC tumors were confirmed and multi-foci metastasis in the lung were seen. The mRNA and protein expression levels of the angiogenesis factors increased in the early stage and as the tumor grew, their expression level enhanced dramatically. Conclusion: The 4T1 syngeneic mouse tumor may serve as an appropriate TNBC model for further investigation of the angiogenesis and therapies. Moreover, angiogenesis factors are induced before the advanced stage, and anti-angiogenesis therapy is necessary to be considered at the first line of treatment in TBNC. [ABSTRACT FROM AUTHOR]

Proposed mechanism for anti-psoriatic activity of GEN on IMQ-induced psoriasis-skin lesions in mice. GEN treatment down-regulates the expression of VEGFR1 and VEGFR2, and suppresses the subsequent phosphorylation of p38 MAPK, leading to the inhibition of pro-angiogenic factor and inflammatory cytokine production, such as VEGF-A, Ang-2, TNF-α, and IL-17A, which at least partly is associated with anti-angiogenic activity of GEN and contributes to the improvement of IMQ-induced psoriasis-like skin lesions in mice. [Display omitted] • For the first time, the protective effect of geniposide (GEN) on imiquimod (IMQ)-induced psoriasis-like skin lesions in mice was investigated. • GEN treatment significantly alleviated the psoriatic skin lesions. • GEN suppressed angiogenesis-related microvascular density and decreased the production of angiogenic facts. • VEGFR1 and VEGFR2 participate in the anti-angiogenesis of GEN in IMQ-induced psoriasis-like skin lesions in mice. In this study, we aimed to evaluate the protective effect of geniposide (GEN) on imiquimod (IMQ)-induced psoriasis-like skin lesions in mice. Firstly, visual changes of psoriatic skin lesions were observed and the severity was recorded using psoriasis area and severity index (PASI) score. Histological changes were assessed by HE staining for epidermal thickness and Masson's staining for collagen fibers. Then, photographs of microvascular inside the skin were taken for macroscopic observation, and microscopic changes associated with angiogenesis were evaluated. Furthermore, expression of angiogenic factors were analyzed by ELISA, immunohistochemistry and immunofluorescence, separately. Lastly, the expression of VEGFR signaling-related proteins was detected by WB. Compared with control, IMQ drove a significant increment of epidermal thicknesses with higher PASI scores and more dermal collagen deposition. IMQ treatment led to abnormal keratinocyte proliferation, increased microvascular inside skin, growing production of angiogenesis-related factors, up-regulated expression of VEGFR1 and VEGFR2, and enhanced phosphorylation of p38. However, GEN significantly ameliorated the psoriatic skin lesions, the epidermal thickness, the formation of collagen fibers, and abnormal keratinocyte proliferation. Importantly, GEN inhibited angiogenesis, the production of angiogenic factors (VEGF-A, Ang-2, TNF-α, and IL-17A), and the proliferation of vascular endothelial cells. Simultaneously, GEN curbed the expression of VEGFR1, VEGFR2, p38, and P-p38 proteins involved in VEGFR signaling. Of note, the suppressive effect of GEN was reversed in the HUVECs with over-expressed VEGFR1 or VEGFR2 related to the cells without transfection. These findings suggest that VEGFR1 and VEGFR2 participate in the anti-angiogenesis of GEN in IMQ-induced psoriasis-like skin lesions in mice. [ABSTRACT FROM AUTHOR]

Aim. To assess the significance of gene expression of the vascular endothelial growth factor-A (VEGF-A) and its interacting receptors VEGFR1 and VEGFR2 as potential diagnostic and prognostic molecular markers in patients with myelodysplastic syndrome (MDS). Materials and methods. A real time polymerase chain reaction (RT-PCR) assay was used to investigate the gene expression of VEGF-A, VEGFR1, and VEGFR2 in the mononuclear cell fractions obtained from 24 patients with MDS. Results. The expression of the 3 genes was identified in all the patients examined. There was the highest expression level of the VEGF-A gene (p

Tumor angiogenesis allows tumor cells to grow and migrate toward the bloodstream and initiate metastasis. The interactions of vascular endothelial growth factors (VEGF) A and B, as the important regulating factors for blood vessel growth, with VEGFR1 and VEGFR2 trigger angiogenesis process. Thus, preventing these interactions led to the effective blockade of VEGF/VEGFRs signaling pathways. In this study, the inhibitory effect of a 23-mer linear peptide (VGB4), which binds to both VEGFR1 and VEGFR2, on VEGF-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) and highly metastatic human breast cancer cell MDA-MB-231 proliferation was examined using MTT assay. To assess the anti-migratory potential of VGB4, HUVECs and also MDA-MB-231 cells wound healing assay was carried out at 48 and 72 h. In addition, downstream signaling pathways of VEGF associated with cell migration and invasion were investigated by quantification of mRNA and protein expression using real-time quantitative PCR and western blot in 4T1 tumor tissues and MDA-MB-231 cells. The results revealed that VGB4 significantly impeded proliferation of HUVECs and MDA-MB-231 cells, in a dose- and time-dependent manner, and migration of HUVECs and MDA-MB-231 cells for a prolonged time. We also observed statistically significant reduction of the transcripts and protein levels of focal adhesion kinase (FAK), Paxillin, matrix metalloproteinase-2 (MMP-2), RAS-related C3 botulinum substrate 1 (Rac1), P21-activated kinase-2 (PAK-2) and Cofilin-1 in VGB4-treated 4T1 tumor tissues compared to controls. The protein levels of phospho-VEGFR1, phospho-VEGFR2, Vimentin, β-catenin and Snail were markedly decreased in both VGB4-treated MDA-MB-231 cells and VGB4-treated 4T1 tumor tissues compared to controls as evidenced by western blotting. These results, in addition to our previous studies, confirm that dual blockage of VEGFR1 and VEGFR2, due to the inactivation of diverse signaling mediators, effectively suppresses tumor growth and metastasis. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is the primary cause of blindness in advanced countries. Repeated intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents has represented an important advancement for the therapy of wet AMD with significative results in terms of blindness prevention and partial vision restore. Nonetheless, some patients are not responsive or do not attain significant visual improvement, intravitreal injection may cause serious complications and important side effects have been reported for the prolonged block of VEGF-A. In order to evaluate new anti-angiogenic strategies, we focused our attention on VEGF receptor 1 (VEGFR1) developing a specific VEGFR-1 antagonist, a tetrameric tripeptide named inhibitor of VEGFR 1 (iVR1). We have evaluated its anti-angiogenic activity in the preclinical model of AMD, the laser-induced choroid neovascularization (CNV). iVR1 is able to potently inhibit CNV when delivered by intravitreal injection. Surprisingly, it is able to significantly reduce CNV also when delivered by gavage. Our data show that the specific block of VEGFR1 in vivo represents a valid alternative to the block of VEGF-A and that the inhibition of the pathological neovascularization at ocular level is also possible by systemic delivery of compounds not targeting VEGF-A. [ABSTRACT FROM AUTHOR]

Aim: To quantify the expression of angiogenic growth factors (ANG2, VEGFA, TGFß1) and their corresponding receptors (VEGFR1, VGFR2, NRP1 and TGFßR1) in human dental pulps from extracted third molars with complete and incomplete root development. Methodology: Fifty‐six dental pulp samples obtained from freshly extracted human third molars were divided equally into two groups according to their stage of root development; 28 third molars with complete root development and 28 third molars with incomplete root development. All samples were processed and total RNA was extracted, cDNA was then synthetized for each sample and the target genes expression profiles for ANG2, VEGFA, VEGFR1, VEGFR2, NRP1, TGFß1 and TGFßR1 were obtained by RT2‐PCR. The data was analysed with a Student's t‐test to compare the replicate ∆∆Ct values for each gene. Results: Teeth with incomplete root development were associated with a significantly greater gene expression of TGFβR1 (P = 0.03), whereas in teeth with complete root development the genes that had significantly greater expression were VEGFA (P = 0.04). Conclusion: The angiogenic growth factors (ANG2, VEGFA, TGFβ1) and their receptors (NRP1, VEGFR1, VEGFR2 and TGFßR1) were expressed in pulps of teeth with complete and incomplete root development measured by RT2‐PCR, with TGFBR1 genes being significantly different in teeth with incomplete root development and VEGFA genes in teeth with complete root development. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor receptor 1 (VEGFR1) reduces the angiogenic activity of vascular endothelial growth factor (VEGF), acting like decoy receptor for VEGF and limiting its availability for genuine angiogenic receptors. The purpose of this study was to establish the significance of VEGFR1 expression in high grade T1 (HGT1) bladder cancer with concomitant carcinoma in situ (CIS) and to determine possible immunohistochemical marker helpful in the follow-up of "unpredictable" HGT1 bladder cancer patients. The analysis included 137 HGT1 bladder cancer samples. Concomitant CIS was diagnosed in 21 (15.33%) of these patients. Sections of 137 formalin-fixed, paraffin-embedded materials were incorporated in tissue microarrays and then stained with a rabbit monoclonal antibody against VEGFR1 (N-term: Y103/- Epitomics, diluted 1:250). Immunohistochemical reaction was scored as following: negative if = 10% of cells were stained and positive if > 10% were stained. We considered both membranous and cytoplasmic expression and staining intensity was scored using a scale of 0 to 3 (0, no staining; 1, weak; 2, moderate; and 3, intense). After a mean follow-up of 50 months, in 137 patients diagnosed with HGT1 urothelial bladder cancer, we found that patients who had concomitant CIS had worse overall survival (p < 0.05), furthermore, those tumour samples had weakly expressed VEGFR1 (p < 0.05). Patients with positive VEGFR1 had longer disease-free (p < 0.01) and overall survival (p < 0.01). Present investigation has revealed that the estimation of VEGFR1 expression could be diagnostic supplement, selecting the HGT1 bladder cancer patients that would require more intensive follow-up, especially if accompanied with CIS. [ABSTRACT FROM AUTHOR]

Incomplete radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) could initiate malignant transition. Patient-derived xenograft (PDX) mice model was established to investigate the effect of VEGF pathway in incomplete RFA of HCC with high fidelity. Cancer stem cell markers and metastatic markers were increased after incomplete RFA, with increased VEGFR1 and decreased VEGFR2 expression. In vitro experiments revealed sublethal heat treatment promoted migration ability of HepG2, HCCLM3, and SMMC7721 cells, which coincided with enhanced ability of sphere formation and up-regulation of VEGFR1, CD133, CD44, and EpCAM. Moreover, HCC cells secreted more VEGF after heat-treatment. VEGF promoted migration and enhanced stemness of HCC cells, which could not be suppressed by VEGFR2 inhibitor. PIGF, the ligand of VEGFR1, significantly increased migration and stemness of HCC cells. Blocking VEGFR1 reduced heat-induced enhancement of migration and stemness, whereas inhibition of VEGFR2 could not. In conclusion, VEGFR1 plays a critical role in sublethal heat treatment-induced enhancement of migration and stemness in HCC, suggesting that VEGFR1 may serve as a potential and promising therapeutic target for preventing recurrence after RFA. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) has long been connected to the development of tissue lesion following ischemic stroke. Contradictory findings either situate VEGF as a promoter of large infarct volumes or as a potential attenuator of damage due to its well documented neuroprotective capability. The core of this discrepancy mostly lies on the substantial number of pleiotropic functions driven by VEGF. Mechanistically, these effects are activated through several VEGF receptors for which various closely related ligands exist. Here, we tested in an experimental model of stroke how the differential activation of VEGF receptors 1 and 2 would modify functional and histological outcomes in the acute phase post-ischemia. We also assessed whether VEGF-mediated responses would involve the modulation of inflammatory mechanisms and how this trophic factor acted specifically on neuronal receptors. We produced ischemic infarcts in adult rats by transiently occluding the middle cerebral artery and induced the pharmacological inhibition of VEGF receptors by i.c.v. administration of the specific VEGFR2 inhibitor SU1498 and the pan-VEGFR blocker Axitinib. We evaluated the neurological performance of animals at 24 h following stroke and the occurrence of brain infarctions analyzed at the gross metabolic and neuronal viability levels. We also assessed the induction of peripheral pro- and anti-inflammatory cytokines in the cerebrospinal fluid and blood and assessed the polarization of activated microglia. Finally, we studied the direct involvement of cortical neuronal receptors for VEGF with in vitro assays of excitotoxic damage. Preferential VEGFR1 activation by the endogenous ligand promotes neuronal protection and prevents the presentation of large volume infarcts that highly correlate with neurological performance, while the concomitant activation of VEGFR2 reduces this effect, even in the presence of exogenous ligand. This process partially involves the polarization of microglia to the state M2. At the cellular level, neurons also responded better to the preferential activation of VEGFR1 when challenged to N -methyl-D-aspartate-induced excitotoxicity. Endogenous activation of VEGFR2 hinders the neuroprotective mechanisms mediated by the activation of VEGFR1. The selective modulation of these concurrent processes might enable the development of therapeutic approaches that target specific VEGFR1-mediated signaling during the acute phase post-stroke. [ABSTRACT FROM AUTHOR]

OBJECTIVES Vascular endothelial growth factor (VEGF) and its receptors play important roles in angiogenesis. Melatonin plays an important role in gonadal development; thus, its effect on the reproductive system is evident. We investigated the influence of melatonin on the expression of VEGF, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), as well as on changes in oxidative stress markers and follicle numbers in rat ovaries. METHODS For this purpose, 45 Wistar rats were separated into the following groups: Group 1, control; Group 2, vehicle; and Group 3, melatonin. Rats in Group 3 were treated with melatonin at 50 mg/kg/day for 30 days. The effects of melatonin on the expression of VEGF, VEGFR1 and VEGFR2 were established by immunohistochemistry analysis. The effects of melatonin on antioxidant enzyme activities were demonstrated by spectrophotometric analysis. RESULTS Based on immunohistochemistry analysis, VEGFR2 was predominantly localized to theca cells in the ovary. Our data indicate that melatonin treatment can significantly increase VEGF and VEGFR1 expression in the ovary ( p

Sprouting angiogenesis is a necessary process in regeneration and development as well as in tumorigenesis. VEGF-A is the main pro-angiogenic chemoattractant and it can bind to the decoy receptor VEGFR1 or to VEGFR2 to induce sprouting. Active sprout cells express Dll4, which binds to Notch1 on neighboring cells, in turn inhibiting VEGFR2 expression. It is known that the balance between VEGFR2 and VEGFR1 determines tip selection and network architecture, however the quantitative interrelationship of the receptors and their interrelated balances, also with relation to Dll4-Notch1 signaling, remains yet largely unknown. Here, we present an agent-based computer model of sprouting angiogenesis, integrating VEGFR1 and VEGFR2 in a detailed model of cellular signaling. Our model reproduces experimental data on VEGFR1 knockout. We show that soluble VEGFR1 improves the efficiency of angiogenesis by directing sprouts away from existing cells over a wide range of parameters. Our analysis unravels the relevance of the stability of the active notch intracellular domain as a dominating hub in this regulatory network. Our analysis quantitatively dissects the regulatory interactions in sprouting angiogenesis. Because we use a detailed model of intracellular signaling, the results of our analysis are directly linked to biological entities. We provide our computational model and simulation engine for integration in complementary modeling approaches.

AIM: To establish the mice model of streptozotocin(STZ)-induced proliferative diabetic retinopathy(PDR), and observe the altered expression of some pro-angiogenic molecules such as vascular endothelial growth factor(VEGF)and its receptors(VEGFR1 and VEGFR2), and matrix metalloproteinase(MMP2 and MMP9)during the development of PDR.METHODS:C57BL/6J mice were intraperitoneal injected with STZ(55 mg/kg)for 5 consecutive days, and blood glucose concentrations were measured after 7d of the injection. The diabetic mice were further housed for 3, 4, 5mo respectively after the development of diabetes. Histological evaluation of retinas was performed. The retinal vessels were detected by immunofluorescence staining with the cluster of differentiation 31(CD31). The mRNA expression of VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 in mice retinas was detected by Real-time PCR analysis.RESULTS: Retinal histological observation and CD31 staining both demonstrate that there are more vessels in diabetic mice than in normal control mice at 5mo after the development of diabetes. As compared with normal control, the mRNA expression of VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 are all increased in diabetic mice at 5mo after the development of diabetes.CONCLUSION: This study demonstrates that PDR is occurred at 5mo after the development of diabetes in STZ-induced diabetic mice. In addition, the mRNA expression of VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 are all increased after the development of PDR.