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Abstract Background Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm infants, characterised by compromised alveolar development and pulmonary vascular abnormalities. Emerging evidence suggests that regulatory T cells (Tregs) may confer protective effects on the vasculature. Knockdown of their transcription factor, interferon regulatory factor 4 (IRF4), has been shown to promote vascular endothelial hyperplasia. However, the involvement of Tregs and IRF4 in the BPD pathogenesis remains unclear. This study aimed to investigate the regulation of Tregs by IRF4 and elucidate its potential role in pulmonary vasculature development in a BPD mouse model. Methods The BPD model was established using 85% hyperoxia exposure, with air exposure as the normal control. Lung tissues were collected after 7 or 14 days of air or hyperoxia exposure, respectively. Haematoxylin–eosin staining was performed to assess lung tissue pathology. Immunohistochemistry was used to measure platelet endothelial cell adhesion molecule-1 (PECAM-1) level, flow cytometry to quantify Treg numbers, and Western blot to assess vascular endothelial growth factor (VEGFA), angiopoietin-1 (Ang-1), forkhead box protein P3 (FOXP3), and IRF4 protein levels. We also examined the co-expression of IRF4 and FOXP3 proteins using immunoprecipitation and immunofluorescence double staining. Furthermore, we employed CRISPR/Cas9 technology to knock down the IRF4 gene and observed changes in the aforementioned indicators to validate its effect on pulmonary vasculature development in mice. Results Elevated IRF4 levels in BPD model mice led to FOXP3 downregulation, reduced Treg numbers, and impaired pulmonary vascular development. Knockdown of IRF4 resulted in improved pulmonary vascular development and upregulated FOXP3 level. Conclusion IRF4 may affect the protective role of Tregs in the proliferation of pulmonary vascular endothelial cells and pulmonary vascular development in BPD model mice by inhibiting the FOXP3 level.

Background: Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm infants, characterised by compromised alveolar development and pulmonary vascular abnormalities. Emerging evidence suggests that regulatory T cells (Tregs) may confer protective effects on the vasculature. Knockdown of their transcription factor, interferon regulatory factor 4 (IRF4), has been shown to promote vascular endothelial hyperplasia. However, the involvement of Tregs and IRF4 in the BPD pathogenesis remains unclear. This study aimed to investigate the regulation of Tregs by IRF4 and elucidate its potential role in pulmonary vasculature development in a BPD mouse model. Methods: The BPD model was established using 85% hyperoxia exposure, with air exposure as the normal control. Lung tissues were collected after 7 or 14 days of air or hyperoxia exposure, respectively. Haematoxylin–eosin staining was performed to assess lung tissue pathology. Immunohistochemistry was used to measure platelet endothelial cell adhesion molecule-1 (PECAM-1) level, flow cytometry to quantify Treg numbers, and Western blot to assess vascular endothelial growth factor (VEGFA), angiopoietin-1 (Ang-1), forkhead box protein P3 (FOXP3), and IRF4 protein levels. We also examined the co-expression of IRF4 and FOXP3 proteins using immunoprecipitation and immunofluorescence double staining. Furthermore, we employed CRISPR/Cas9 technology to knock down the IRF4 gene and observed changes in the aforementioned indicators to validate its effect on pulmonary vasculature development in mice. Results: Elevated IRF4 levels in BPD model mice led to FOXP3 downregulation, reduced Treg numbers, and impaired pulmonary vascular development. Knockdown of IRF4 resulted in improved pulmonary vascular development and upregulated FOXP3 level. Conclusion: IRF4 may affect the protective role of Tregs in the proliferation of pulmonary vascular endothelial cells and pulmonary vascular development in BPD model mice by inhibiting the FOXP3 level. [ABSTRACT FROM AUTHOR]

Abstract Background Hypoxic pulmonary hypertension (HPH) is a common type of pulmonary hypertension and characterized by pulmonary vascular remodeling and constriction. A large number of studies have shown that pulmonary vascular endothelial cells (PVECs) dysfunction plays an important role in the initiation and development stages of HPH, but the mechanism of PVECs dysfunction after hypoxia remains unclear. In this study, we explored the exact mechanism of PVECs dysfunction after hypoxia. Methods In vitro, we used primary cultured PVECs hypoxia model to mimic HPH injury. We detected the expressions of mitochondrial biogenesis markers, mitochondrial transcription factor A (TFAM) level inside mitochondria, mitochondrial quantity and function, and the components expressions of translocase of outer mitochondrial membrane (TOM) at 24 h after hypoxia. To explore the effects of Tom70 on mitochondrial biogenesis and functions of PVECs after hypoxia, Tom70 overexpression adenovirus was constructed, and the expressions of mitochondrial biogenesis markers, TFAM level inside mitochondria, mitochondrial quantity and function, and the functions of PVECs were detected. And in vivo, we used cre-dependent overexpression adenovirus of Tom70 in the Cdh5-CreERT2 mouse model of HPH to verify the role of upregulating PVECs Tom70 in improving HPH. Results Hypoxia obviously increased the expressions of mitochondrial biogenesis markers for PGC-1α, NRF-1 and TFAM, but reduced the content of TFAM in mitochondria and the quantity and functions of mitochondria. In addition, only Tom70 expression among the TOM components was significantly decreased after hypoxia, and up-regulation of Tom70 significantly increased the content of TFAM in mitochondria of PVECs by transporting TFAM into mitochondria after hypoxia, enhanced the quantity and functions of mitochondria, improved the functions of PVECs, and ultimately alleviated HPH. Conclusion The findings of present study demonstrated that hypoxia induced the decreased expression of Tom70 in PVECs, reduced the mitochondrial biogenesis-associated TFAM protein transporting into mitochondria, inhibited mitochondrial biogenesis, caused PVECs injury, and prompted the formation of HPH. However, up-regulation of Tom70 abolished the hypoxia-induced injurious effects on PVECs and alleviated HPH.

Background: Hypoxic pulmonary hypertension (HPH) is a common type of pulmonary hypertension and characterized by pulmonary vascular remodeling and constriction. A large number of studies have shown that pulmonary vascular endothelial cells (PVECs) dysfunction plays an important role in the initiation and development stages of HPH, but the mechanism of PVECs dysfunction after hypoxia remains unclear. In this study, we explored the exact mechanism of PVECs dysfunction after hypoxia. Methods: In vitro, we used primary cultured PVECs hypoxia model to mimic HPH injury. We detected the expressions of mitochondrial biogenesis markers, mitochondrial transcription factor A (TFAM) level inside mitochondria, mitochondrial quantity and function, and the components expressions of translocase of outer mitochondrial membrane (TOM) at 24 h after hypoxia. To explore the effects of Tom70 on mitochondrial biogenesis and functions of PVECs after hypoxia, Tom70 overexpression adenovirus was constructed, and the expressions of mitochondrial biogenesis markers, TFAM level inside mitochondria, mitochondrial quantity and function, and the functions of PVECs were detected. And in vivo, we used cre-dependent overexpression adenovirus of Tom70 in the Cdh5-CreERT2 mouse model of HPH to verify the role of upregulating PVECs Tom70 in improving HPH. Results: Hypoxia obviously increased the expressions of mitochondrial biogenesis markers for PGC-1α, NRF-1 and TFAM, but reduced the content of TFAM in mitochondria and the quantity and functions of mitochondria. In addition, only Tom70 expression among the TOM components was significantly decreased after hypoxia, and up-regulation of Tom70 significantly increased the content of TFAM in mitochondria of PVECs by transporting TFAM into mitochondria after hypoxia, enhanced the quantity and functions of mitochondria, improved the functions of PVECs, and ultimately alleviated HPH. Conclusion: The findings of present study demonstrated that hypoxia induced the decreased expression of Tom70 in PVECs, reduced the mitochondrial biogenesis-associated TFAM protein transporting into mitochondria, inhibited mitochondrial biogenesis, caused PVECs injury, and prompted the formation of HPH. However, up-regulation of Tom70 abolished the hypoxia-induced injurious effects on PVECs and alleviated HPH. [ABSTRACT FROM AUTHOR]

AIM: To study the function of interferon regulatory factor 4 (IRF4) in the lung development of bronchopulmonary dysplasia (BPD) model mice by analyzing the interaction of IRF4 and forkhead box P3 (FOXP3) and their relevance with the proliferation of pulmonary vascular endothelial cells (PVECs). METHODS: The BPD model of C57BL/6 mice exposed to 85% hyperoxia was built, and the mice in normoxia group were used as normal control. These mice were randomly divided into normoxia for 7 d (N7) group, hyperoxia for 7 d (H7) group, normoxia for 14 d (N14) group and hyperoxia for 14 d (H14) group. The lung tissue was obtained at 7 and 14 d after exposure to normoxia or hyperoxia, and the pathological changes of lung tissues were observed by hematoxylin-eosin staining. The expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a proliferative marker of PVECs, was detected by immunohistochemical staining. Western blot was used to detect the protein expression levels of vascular endothelial growth factor A (VEGFA), angiopoietin-1 (Ang-1), FOXP3 and IRF4. The interaction of IRF4 and FOXP3 proteins was detected by co-immunoprecipitation. RESULTS: Compared with normoxia group simultaneously, the average optical density of CD31 positive cells and the density of pulmonary vessels in hyperoxia group were significantly decreased (P<0. 01). The expression of VEGFA, Ang-1 and FOXP3 proteins in the lung tissue of mice in hyperoxia group was substantially reduced (P< 0. 01). Co-immunocoprecipitation showed that there was association between IRF4 and FOXP3, and the correlation of hyperoxia group was significantly enhanced compared with the normoxia group (P<0. 01). The expression of IRF4 and FOXP3 protein was significantly negatively correlated (R=−0. 932, P<0. 01), and the expression of FOXP3 protein was substantially positively correlated with CD31 (R=0. 865, P<0. 01). CONCLUSION: IRF4 may impact the proliferation of PVECs and the development of pulmonary vessels by inhibiting the expression of FOXP3 protein in the hyperoxia-induced BPD model mice. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is the leading cause of bacterial sepsis-related death because of disrupted pulmonary endothelial barrier, resulting in protein-rich pulmonary oedema, an influx of pro-inflammatory cells and refractory hypoxaemia. Several studies have reported that C3a levels are significantly higher in organs with sepsis and their peripheral organs and are closely associated with organ dysfunction and poor prognosis in sepsis. However, the role of the C3a complement in sepsis ALI remains unclear. Therefore, this study aimed to investigate the important role and mechanism of C3a in preventing the occurrence of pyroptosis (a pro-inflammatory form of cell death) to protect the lung endothelial cells (ECs) in sepsis-induced ALI. A septic mouse model was established with cecal ligation and puncture (CLP), which demonstrated that C3a mediated EC pyroptosis through its C3aR receptor. Furthermore, inhibition of the C3a–C3aR axis could block both NLRP3/caspase-1 and caspase-11 pathways, thus preventing pulmonary EC from pyroptosis. These results indicate that inhibition of the C3A–C3AR complement axis can inhibit pulmonary vascular EC pyroptosis, a potential target for the treatment of ALI. [Display omitted] • The expression of C3/C3a and C3aR is increased in septic mice. • C3a-C3aR blockage can inhibit the lung ECs pyroptosis and improve ALI in CLP mic. • C3a-C3aR protects ALI by suppressing the NLRP3/caspase-1 and caspase-11. [ABSTRACT FROM AUTHOR]

In the lung, communication between pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) is essential for the maintenance of vascular homeostasis. In pulmonary hypertension (PH), the derangement in their cell–cell communication plays a major role in the pathogenesis of pulmonary vascular remodeling. In this study, we focused on the role of PVEC‐derived extracellular vesicles (EV), specifically their microRNA (miRNA, miR‐) cargo, in the regulation of PASMC proliferation and vascular remodeling in PH. We found that the amount of pro‐proliferative miR‐210‐3p was increased in PVEC‐derived EV in hypoxia (H‐EV), which contributes to the H‐EV‐induced proliferation of PASMC and the development of PH. [ABSTRACT FROM AUTHOR]

Abstract In the lung, communication between pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) is essential for the maintenance of vascular homeostasis. In pulmonary hypertension (PH), the derangement in their cell–cell communication plays a major role in the pathogenesis of pulmonary vascular remodeling. In this study, we focused on the role of PVEC‐derived extracellular vesicles (EV), specifically their microRNA (miRNA, miR‐) cargo, in the regulation of PASMC proliferation and vascular remodeling in PH. We found that the amount of pro‐proliferative miR‐210‐3p was increased in PVEC‐derived EV in hypoxia (H‐EV), which contributes to the H‐EV‐induced proliferation of PASMC and the development of PH.

Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0–25 uM) and medium (25–50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent.

The present study aimed to evaluate the function of pyrin and interleukin-10 (IL-10) and the potential mechanisms underlying the regulation of inflammation in pulmonary vascular endothelial cells (ECs) following hemorrhagic shock (HS). Adult female Sprague-Dawley rats were divided into 4 groups (n=6 in each group) to examine the changes in pyrin expression following HS-lipopolysaccharide (LPS) administration, including the following groups: A sham operation (SM) + tracheal injection of saline (SAL) group; a HS + SAL group; a SM + LPS group (with a tracheal injection of endotoxin); and a HS + LPS group. An additional 4 groups were used to evaluate the function of IL-10, by the additional intratracheal injection of recombinant IL-10. Western blot analysis and immunofluorescence were performed in order to investigate the changes to pyrin and IL-10 expression in pulmonary vascular ECs. The expression levels of pyrin in the SM + LPS group were significantly increased in comparison with the SM + SAL group (P<0.01). Additionally, the expression levels of pyrin were significantly increased in the HS + LPS group compared with the HS + SAL group (P<0.01). The expression levels of caspase-1 were significantly increased in the HS + LPS group compared with those in the other three groups (P<0.01). The expression levels of pyrin in the HS + LPS + IL-10 group were significantly increased compared with the HS + LPS group (P<0.01). The expression levels of caspase-1 were significantly decreased following IL-10 treatment compared with those in the HS + LPS group (P<0.01). Therefore, HS attenuated LPS-induced pyrin expression in pulmonary vascular ECs and may also inhibit the expression of IL-10, resulting in the activation of caspase-1 subsequent to a second LPS insult. [ABSTRACT FROM AUTHOR]

Abstract Hepatopulmonary syndrome (HPS) is a serious complication in patients with advanced liver disease. The pathological pulmonary angiogenesis contributes to the progression of HPS. Importantly, directional collective migration of endothelial cells is a critical event for pathological angiogenesis. Previously, we have demonstrated that the over-expression of Cyclooxygenase-2 (COX-2) was an important factor in the experimental HPS. However, the role of COX-2 in the directional collective migration of human pulmonary microvascular endothelial cells (HPMVECs) is unclear. Our study aims to evaluate the potential effect of COX-2 in the directional collective migration of HPMVECs under the stimulation of HPS patient serum. In this study, 9 patients with stable liver cirrhosis were screened for presence of HPS. We confirmed that HPS patient serum dramatically promoted the directional collective migration and angiogenesis of HPMVECs, while the COX-2 selective antagonist parecoxib significantly inhibited the directional collective migration of HPMVEC under the stimulation of HPS patient serum. In addition, HPS patient serum significantly upregulated the phosphorylation of PKC and promoted the activation of Rac via COX-2/PGE2 signaling pathway. Notably, silencing PKC activation attenuated the directional collective migration of HPMVEC induced by HPS patient serum. In conclusion, these results indicate that PKC/Rac signaling induced by COX-2 modulates collective directional migration of HPMVEC during pathological pulmonary angiogenesis in HPS patients. Highlights • HPS patient serum promotes the directional collective migration of HPMVECs. • COX-2/PGE2 induces directional collective migration of HPMVECs under HPS patient serum stimulation via PKC/Rac signaling. • COX-2 selective inhibitor may be a hopeful treatment for pathological pulmonary angiogenesis in HPS patients. [ABSTRACT FROM AUTHOR]

Pulmonary vascular endothelial cells could contribute to maintain homeostasis in adult lung vasculature. "Tissue-resident" endothelial progenitor cells (EPCs) play pivotal roles in postnatal vasculogenesis, vascular repair, and tissue regeneration; however, their local pulmonary counterparts remain to be defined. To determine whether prominin-1/CD133 expression can be a marker of tissue-resident vascular EPCs in the pulmonary circulation, we examined the origin and characteristics of prominin-1/CD133-positive (Prom1+) PVECs considering cell cycle status, viability, histological distribution, and association with pulmonary vascular remodeling. Prom1+ PVECs exhibited high steady-state transit through the cell cycle compared with Prom1- PVECs and exhibited homeostatic cell division as assessed using the label dilution method and mice expressing green fluorescent protein. In addition, Prom1+ PVECs showed more marked expression of putative EPC markers and drug resistance genes as well as highly increased activation of aldehyde dehydrogenase compared with Prom1- PVECs. Bone marrow reconstitution demonstrated that tissue- resident cells were the source of >98% of Prom1+ PVECs. Immunofluorescence analyses revealed that Prom1+ PVECs preferentially resided in the arterial vasculature, including the resistant vessels of the lung. The number of Prom1+ PVECs was higher in developing postnatal lungs. Sorted Prom1+ PVECs gave rise to colonies and formed fine vascular networks compared with Prom1- PVECs. Moreover, Prom1+ PVECs increased in the monocrotaline and the Su-5416 + hypoxia experimental models of pulmonary vascular remodeling. Our findings indicated that Prom1- PVECs exhibited the phenotype of tissue-resident EPCs. The unique biological characteristics of Prom1- PVECs predominantly contribute to neovasculogenesis and maintenance of homeostasis in pulmonary vascular tissues. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) can activate endothelial cells and induce inflammatory injury. Toll-like receptor-4 (TLR-4) is integrally involved in LPS signaling and has a requisite role in the activation of nuclear factor (NF)-κB. A number of studies have demonstrated the cytoprotective action of perfluorocarbon (PFC) both in vivo and in vitro, but the exact mechanisms have yet to be elucidated. In this study, we examined in an in vitro model the cytoprotective effect of PFC on LPS-stimulated pulmonary vascular endothelial cells (PMVECs). Intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) were significantly increased in the LPS-stimulated PMVECs groups. The expression of TLR-4 mRNA and protein in LPS groups was markedly increased. Meanwhile, NF-κB was activated. There were no significant effects of PFC alone on any of the factors studied while the coculture group showed significant downregulation of the secretion of ICAM-1, TNF-α, and IL-8; the expression of TLR-4 mRNA; and the activity of NF-κB. LPS can induce PMVEC inflammatory injury via the activation of TLR-4 and subsequent activation of NF-κB. PFC is able to protect PMVECs from LPS-induced inflammatory injury by blocking the initiation of the LPS signaling pathway. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a progressive lung disease caused by thickening of the pulmonary arterial wall and luminal obliteration of the small peripheral arteries leading to increase in vascular resistance which elevates pulmonary artery pressure that eventually causes right heart failure and death. We have previously shown that transcription factor Msx1 (mainly expressed during embryogenesis) is strongly upregulated in transformed lymphocytes obtained from PAH patients, especially IPAH. Under pathological conditions, Msx1 overexpression can cause cell dedifferentiation or cell apoptosis. We hypothesized that Msx1 overexpression contributes to loss of small pulmonary vessels in PAH. In IPAH lung, MSX1 protein localization was strikingly increased in muscularized remodeled pulmonary vessels, whereas it was undetectable in control pulmonary arteries. We developed a transgenic mouse model overexpressing MSX1 (MSX1OE) by about 4-fold and exposed these mice to normoxic, sugen hypoxic (3 weeks) or hyperoxic (100% 02 for 3 weeks) conditions. Under normoxic conditions, compared to controls, MSX1OE mice demonstrated a 30-fold and 2-fold increase in lung Msx1 mRNA and protein expression, respectively. There was a significant retinal capillary dropout (p < 0.01) in MSX1OE mice, which was increased further (p < 0.03) with sugen hypoxia. At baseline, the number of pulmonary vessels in MSX1OE mice was similar to controls. In sugen-hypoxia-treated MSX1OE mice, the number of small (0–25 uM) and medium (25–50 uM) size muscularized vessels increased approximately 2-fold (p < 0.01) compared to baseline controls; however, they were strikingly lower (p < 0.001) in number than in sugen-hypoxia-treated control mice. In MSX1OE mouse lung, 104 genes were upregulated and 67 genes were downregulated compared to controls. Similarly, in PVECs, 156 genes were upregulated and 320 genes were downregulated from siRNA to MSX1OE, and in PVSMCs, 65 genes were upregulated and 321 genes were downregulated from siRNA to MSX1OE (with control in the middle). Many of the statistically significant GO groups associated with MSX1 expression in lung, PVECs, and PVSMCs were similar, and were involved in cell cycle, cytoskeletal and macromolecule organization, and programmed cell death. Overexpression of MSX1 suppresses many cell-cycle-related genes in PVSMCs but induces them in PVECs. In conclusion, overexpression of Msx1 leads to loss of pulmonary vessels, which is exacerbated by sugen hypoxia, and functional consequences of Msx1 overexpression are cell-dependent. [ABSTRACT FROM AUTHOR]

The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce β-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca 2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced β-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (C X C motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Gao et al.)

Severe acute pancreatitis (SAP) is a disease with a high mortality. Patients with SAP may also be complicated with acute lung injury (ALI). So far the therapy for SAP-ALI is still limited. Emerging evidences demonstrate that microRNAs (miRs) could play a role in SAP-ALI. This study aims to define the role of miR-339-3p in SAP-ALI via Anxa3 through the Akt/mTOR signaling pathway. Ten mice were selected as sham group and 36 mice as model group which further assigned into different groups. Relationship between miR-339-3p and Anxa3 was detected by dual luciferase reporter gene assay. Levels of TNF-α, IL-6, and serum amylase (AMS) and myeloperoxidase (MPO) in lung tissues were determined by ELISA. Expression of related genes in pulmonary vascular endothelial cells (PMVECs) and lungs tissues was determined by Western blot analysis and RT-qPCR. Cell apoptosis was detected by flow cytometry and TUNEL. SAP-ALI mice had decreased survival rate, increased levels of TNF-α, IL-6, AMS, MPO, and Schmidt scores. miR-339-3p was poorly expressed in lung tissue of SAP-ALI mice while Anxa3 was reciprocal. Anxa3 was targeted by miR-339-3p. miR-339-3p inhibited the relative expression of the Akt/mTOR signaling pathway-related proteins, alleviated inflammation and edema of SAP-ALI mice, and suppressed apoptosis of PMVECs; Anxa3 exhibited opposite trends. In conclusion, overexpressed miR-339-3p could suppress Anxa3 to inhibit the Akt/mTOR signaling pathway, so as to decrease tissue edema, inflammation, and PMVEC apoptosis in SAP-ALI mice., (© 2018 Wiley Periodicals, Inc.)

Pulmonary vascular endothelial cells could contribute to maintain homeostasis in adult lung vasculature. "Tissue-resident" endothelial progenitor cells (EPCs) play pivotal roles in postnatal vasculogenesis, vascular repair, and tissue regeneration; however, their local pulmonary counterparts remain to be defined. To determine whether prominin-1/CD133 expression can be a marker of tissue-resident vascular EPCs in the pulmonary circulation, we examined the origin and characteristics of prominin-1/CD133-positive (Prom1(+)) PVECs considering cell cycle status, viability, histological distribution, and association with pulmonary vascular remodeling. Prom1(+) PVECs exhibited high steady-state transit through the cell cycle compared with Prom1(-) PVECs and exhibited homeostatic cell division as assessed using the label dilution method and mice expressing green fluorescent protein. In addition, Prom1(+) PVECs showed more marked expression of putative EPC markers and drug resistance genes as well as highly increased activation of aldehyde dehydrogenase compared with Prom1(-) PVECs. Bone marrow reconstitution demonstrated that tissue-resident cells were the source of >98% of Prom1(+) PVECs. Immunofluorescence analyses revealed that Prom1(+) PVECs preferentially resided in the arterial vasculature, including the resistant vessels of the lung. The number of Prom1(+) PVECs was higher in developing postnatal lungs. Sorted Prom1(+) PVECs gave rise to colonies and formed fine vascular networks compared with Prom1(-) PVECs. Moreover, Prom1(+) PVECs increased in the monocrotaline and the Su-5416 + hypoxia experimental models of pulmonary vascular remodeling. Our findings indicated that Prom1(+) PVECs exhibited the phenotype of tissue-resident EPCs. The unique biological characteristics of Prom1(+) PVECs predominantly contribute to neovasculogenesis and maintenance of homeostasis in pulmonary vascular tissues., (Copyright © 2016 the American Physiological Society.)