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Several studies have shown that an abnormal vascular-immunity link could increase Alzheimer’s disease (AD) risk; however, the mechanism is unclear. CD31, also named platelet endothelial cell adhesion molecule (PECAM), is a surface membrane protein of both endothelial and immune cells and plays important roles in the interaction between the vascular and immune systems. In this review, we focus on research regarding CD31 biological actions in the pathological process that may contribute to AD based on the following rationales. First, endothelial, leukocyte and soluble forms of CD31 play multi-roles in regulating transendothelial migration, increasing blood–brain barrier (BBB) permeability and resulting in neuroinflammation. Second, CD31 expressed by endothelial and immune cells dynamically modulates numbers of signaling pathways, including Src family kinases, selected G proteins, and β-catenin which in turn affect cell-matrix and cell–cell attachment, activation, permeability, survival, and ultimately neuronal cell injury. In endothelia and immune cells, these diverse CD31-mediated pathways act as a critical regulator in the immunity-endothelia-brain axis, thereby mediating AD pathogenesis in ApoE4 carriers, which is the major genetic risk factor for AD. This evidence suggests a novel mechanism and potential drug target for CD31 in the background of genetic vulnerabilities and peripheral inflammation for AD development and progression.

Background Atrial myxomas are generally considered benign neoplasms. The majority of tumors are sporadic and less than 10% are associated with an autosomal dominant condition known as the Carney complex, which is most often caused by germline mutation in the gene PRKAR1A. Whether this gene plays a role in the development of sporadic myxomas has been an area of debate, although recent studies have suggested that some fraction of sporadic tumors also carry mutations in PRKARIA. Extra-cardiac complications of atrial myxoma include dissemination of tumor to the brain; however, the dissemination of viable invasive tumor cells is exceedingly rare. Case presentation We present here a 48-year-old white woman who developed multiple intracranial hemorrhagic lesions secondary to tumor embolism that progressed to ‘false’ aneurysm formation and invasion through the vascular wall into brain parenchyma 7 months after resection of an atrial myxoma. Whole exome sequencing of her tumor revealed multiple mutations in PRKAR1A not found in her germline deoxyribonucleic acid (DNA), suggesting that the myxoma in this patient was sporadic. Conclusions Our patient illustrates that mutations in PRKAR1A may be found in sporadic lesions. Whether the presence of this mutation affects the clinical behavior of sporadic tumors and increases risk for metastasis is not clear. Regardless, the protein kinase A pathway which is regulated by PRKAR1A represents a possible target for treatment in patients with metastatic cardiac myxomas harboring mutations in the PRKARIA gene.

Neonatal hypoxic injury (NHI) is a devastating cause of disease that affects >60% of babies born with a very low birth weight, resulting in significant morbidity and mortality, including life-long neurological consequences such as seizures, cerebral palsy, and intellectual disability. Hypoxic injury results in increased neuronal death, which disrupts normal brain development. Although animal model systems have been useful to study the effects of NHI, they do not fully represent the uniqueness and complexities of the human brain. To better understand the effects of hypoxia on human brain development, we have generated a brain organoid protocol and evaluated these cells over the course of 6 months. As anticipated, the expression of a forebrain marker, FOXG1, increased and then remained expressed over time, while there was a transition in the expression of the deep-layer (TBR1) and upper-layer (SATB2) cortical markers. In addition, ventral genes (Eng1 and Nkx2.1) as well as markers of specialized nonneuronal cells (Olig2 and GFAP) also increased at later time points. We next tested the development of our in vitro cerebral organoid model at different oxygen concentrations and found that hypoxia repressed gene markers for forebrain, oligodendrocytes, glial cells, and cortical layers, as well as genes important for the migration of cortical neurons. In contrast, ventral markers were either unaffected or even increased in expression with hypoxic insult. Interestingly, the negative effect of hypoxia on the dorsal brain genes as well as oligodendrocytes, and neuronal progenitors could be mitigated by the use of minocycline, an FDA-approved small molecule. Taken together, we have generated a unique and relevant in vitro human brain model system to study diseases such as NHI as well as their potential treatments. Using this system, we have shown the efficacy of minocycline for human NHI.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid's growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

Neutrophils from NOD (Non-Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild-type [WT] C57BL/6 mice). Additionally, F-actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393 →K) (E372 of LynB), affecting fMLP-induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. J. Cell. Physiol. 232: 1689-1695, 2017. © 2016 Wiley Periodicals, Inc.

Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced protein extraction method using known quantities of proteotypic 13C-labeled peptides to quantify matrix proteins in native and decellularized lung tissues. Using quantitative proteomics that produce picomole-level measurements of a large number of matrix proteins, we show that a mild decellularization technique (“Triton/SDC”) results in near-native retention of laminins, proteoglycans, and other basement membrane and ECM-associated proteins. Retention of these biologically important glycoproteins and proteoglycans is quantified to be up to 27-fold higher in gently-decellularized lung scaffolds compared to scaffolds generated using a previously published decellularization regimen. Cells seeded onto this new decellularized matrix also proliferate robustly, showing positive staining for proliferating cell nuclear antigen (PCNA). The high fidelity of the gently decellularized scaffold as compared to the original lung extracellular matrix represents an important step forward in the ultimate recapitulation of whole organs using tissue-engineering techniques. This method of ECM and scaffold protein analysis allows for better understanding, and ultimately quality control, of matrices that are used for tissue engineering and human implantation. These results should advance regenerative medicine in general, and whole organ regeneration in particular. Statement of Significance The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N-cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here, we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild-type (WT-FB) and CD44 knockout dermal fibroblasts (CD44KO-FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO-FB cultures. Additionally, we found that siRNA knock-down of CD44 mimicked the behaviors of CD44KO-FB, restoring the decreases in N-cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho-YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO-FB. Interestingly, plating CD44KO-FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non-coated dishes, consistent with increased cell adhesion compared to their effects on WT-FB. Lastly, siRNA knockdown of CD44 in WT-FB resulted in increased fibroblast migration compared to WT-FB, albeit at reduced rates compared to CD44KO-FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues.

Microvascular endothelial cells cultured in three-dimensional hydrogel scaffolds form a network of microvessel structures when implanted subcutaneously in mice, inosculate with host vessels, and over time remodel into large ectatic vascular structures resembling hemangiomas. When compared with infantile hemangiomas, similarities were noted, including a temporal progression from a morphological appearance of a proliferative phase to the appearance of an involuted phase, mimicking the proliferative and involutional phases of infantile hemangioma. Consistent with the progression of a proliferative phase to an involuted phase, both the murine implants and human biopsy tissue exhibit reduced expression of Ajuba, YAP, and Survivin labeling as they progressed over time. Significant numbers of CD45+, CD11b+, Mac3+ mononuclear cells were found at the 2-week time point in our implant model that correlated with the presence of CD45+, CD68+ mononuclear cells observed in biopsies of human proliferative-phase hemangiomas. At the 4-week time point in our implant model, only small numbers of CD45+ cells were detected, which again correlated with our findings of significantly diminished CD45+, CD68+ mononuclear cells in human involutional-phase hemangiomas. The demonstration of mononuclear cell infiltration transiently in the proliferative phase of these lesions suggests that the vascular proliferation and/or regression may be driven in part by an immune response. Gross and microscopic morphological appearances of human proliferative and involutional hemangiomas and our implant model correlate well with each other as do the expression levels of Hippo pathway components (Ajuba and YAP) and Survivin and correlate with proliferation in these entities. Inhibitors of Survivin and Ajuba (which we have demonstrated to inhibit proliferation and increase apoptosis in murine hemangioendothelioma cell tissue culture) may have potential as other beneficial treatments for proliferating infantile hemangiomas. This implant model may have potential as a modest through-put screen for testing and development of therapeutics targeted at the proliferative phase of infantile hemangiomas, reducing the subsequent postinvolutional scarring or deformities sometimes associated with these lesions.

Hemangioendotheliomas are categorized as intermediate-grade vascular tumors that are commonly localized in the lungs and livers. The regulation of this tumor cell's proliferative and apoptotic mechanisms is ill defined. We recently documented an important role for Hippo pathway signaling via endothelial cell adhesion molecules in brain microvascular endothelial cell proliferation and apoptosis. We found that endothelial cells lacking cell adhesion molecules escaped from contact inhibition and exhibited abnormal proliferation and apoptosis. Here we report on the roles of adherens junction molecule modulation of survivin and the Hippo pathway in the proliferation and apoptosis of a murine hemangioendothelioma (EOMA) cell. We demonstrated reduced adherens junction molecule (CD31 and VE-cadherin) expression, increased survivin and Ajuba expression, and a reduction in Hippo pathway signaling resulting in increased proliferation and decreased activation of effector caspase 3 in postconfluent EOMA cell cultures. Furthermore, we confirmed that YM155, an antisurvivin drug that interferes with Sp1-survivin promoter interactions, and survivin small interference RNA (siRNA) transfection elicited induction of VE-cadherin, decreased Ajuba expression, increased Hippo pathway and caspase activation and apoptosis, and decreased cell proliferation. These findings support the importance of the Hippo pathway in hemangioendothelioma cell proliferation and survival and YM155 as a potential therapeutic agent in this category of vascular tumors.

Matrix Metalloproteinase2, (MMP2, gelatinase A) is a zinc-containing enzyme with a broad substrate specificity including components of the extracellular matrix, cell surface molecules and a wide range bioactive molecules. MMP2 is known to play important roles in a variety of signaling pathways and processes in a wide range of cell types and tissues. In this report we elucidate the effects of the absence of MMP2 in Neural Precursor Cells (NPC) derived from C57BL/6 MMP2 KO mice and in primary and secondary neurosphere formation. We observed smaller neurosphere numbers and sizes, decreased NPC numbers, PCNA expression, DNA and Akt activation in MMP2 KO NPC compared to WT NPC. We also found decreased neurosphere formation and NPC migration outward from adherent neurospheres, decreased CXCR4 and nestin expression and increased GFAP and neuro-filament expression in MMP2 KO NPC compared to Wt NPC. MMP2 KO mice were found to exhibit increased anxiety manifested in open field activity assays compared to Wt mice. MMP2 KO mice also exhibited differences in motor activities manifested by decreased balance and endurance during Rota-rod testing. These studies illustrate an important role of MMP2 in cognitive and motor behaviors and confirm its importance in NPC activities crucial to brain development, growth and response to and recovery from injury.

CD44 has been implicated in a diverse array of cell behaviors and in a diverse range of signaling pathway activations under physiological and pathophysiological conditions. We have documented a role for CD44 in mediating vascular barrier integrity via regulation of PECAM-1 (CD31) expression. We now report our findings on the roles of CD44 in modulating proliferation and apoptosis of microvascular endothelial cells via its modulation of CD31 and VE-cadherin expression and the Hippo pathway. In this report, we demonstrate persistent increased proliferation and reduced activations of both effector and initiator caspases in high cell density, postconfluent CD44 knock-out (CD44KO), and CD31KO cultures. We found that reconstitution with murine CD44 or CD31 restored the proliferative and caspase activation rates to WT levels. Moreover, we have confirmed that the CD31 ecto-domain plays a key role in specific caspase cascades as well as cell adhesion-mediated cell growth and found that CD31 deficiency results in a reduction in VE-cadherin expression. Last, we have shown that both CD44KO and CD31KO endothelial cells exhibit a reduced VE-cadherin expression correlating with increased survivin expression and YAP nuclear localization, consistent with inactivation of the Hippo pathway, resulting in increased proliferation and decreased apoptosis. These findings support the concept that CD44 mediates several of its effects on endothelia through modulation of adhesion protein expression, which, in addition to its known modulation of junctional integrity, matrix metalloproteinase levels and activation, interactions with cortical membrane proteins, and selected signaling pathways, plays a key role as a critical regulator of vascular function.

Several models of arteriovenous fistula (AVF) have excellent patency and help in understanding the mechanisms of venous adaptation to the arterial environment. However, these models fail to exhibit either maturation failure or fail to develop stenoses, both of which are critical modes of AVF failure in human patients. We used high-resolution Doppler ultrasound to serially follow mice with AVFs created by direct 25-gauge needle puncture. By day 21, 75% of AVFs dilate, thicken, and increase flow, i.e., mature, and 25% fail due to immediate thrombosis or maturation failure. Mature AVF thicken due to increased amounts of smooth muscle cells. By day 42, 67% of mature AVFs remain patent, but 33% of AVFs fail due to perianastomotic thickening. These results show that the mouse aortocaval model has an easily detectable maturation phase in the first 21 days followed by a potential failure phase in the subsequent 21 days. This model is the first animal model of AVF to show a course that recapitulates aspects of human AVF maturation.

Adhesion molecule CD44 is expressed by multiple cell types and is implicated in various cellular and immunological processes. In this study, we examined the effect of global CD44 deficiency on myelin oligodendrocyte glycoprotein peptide (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Compared to C57BL/6 wild-type mice, CD44-deficient mice presented with greater disease severity, increased immune cell numbers in the central nervous system, and increased anti-MOG antibody and proinflammatory cytokine production, especially those associated with T helper 17 (Th17) cells. Further, decreased numbers of peripheral CD4+CD25+FoxP3+ regulatory T cells (Tregs) were observed in CD44-knockout mice throughout the disease course. CD44-knockout CD4 T cells exhibited reduced transforming growth factor-β receptor type I (TGF-β RI) expression that did not impart a defect in Treg polarization in vitro, but did correlate with enhanced Th17 polarization in vitro. Further, EAE in bone marrow–chimeric animals suggested CD44 expression on both circulating and noncirculating cells limited disease severity. Endothelial expression of CD44 limited T-cell adhesion to and transmigration through murine endothelial monolayers in vitro. Importantly, we also identified increased permeability of the blood–brain barrier in vivo in CD44-deficient mice before and following immunization. These data suggest that CD44 has multiple protective roles in EAE, with effects on cytokine production, T-cell differentiation, T-cell–endothelial cell interactions, and blood–brain barrier integrity.

Chronic sublethal hypoxia, a complication of premature birth, is associated with cognitive and motor handicaps. Responsiveness to and recovery from this hypoxic environment is dependent on induction of HIF-1 α in the cells affected. Microvascular endothelial-glial and microvascular endothelial-neuronal precursor interactions have been found to be dynamic and reciprocal, involving autocrine and paracrine signaling, with response and recovery correlated with baseline levels and levels of induction of HIF-1 α.To ascertain the roles of endothelial HIF-1 α in the responses of brain microvascular endothelial cells (EC) and neuronal precursors to hypoxia, we examined the effects of the presence and absence of endothelial HIF-1 α expression in culture and in cells comprising the subventricular zone (SVZ) and dentate gyrus under normoxic and hypoxic conditions. We used C57BL/6 WT and EC HIF-1 α -deficient mice and brain microvascular ECs isolated from these mice in western blots, immunofluorescence, and behavioral studies to examine the roles of EC HIF-1 α behaviors of endothelial and neuronal precursor cells (NPCs) in SVZ and hippocampal tissues under normoxic and hypoxic conditions and behaviors of these mice in open field activity tests. Analyses of ECs and SVZ and dentate gyrus tissues revealed effects of the absence of endothelial HIF-1 α on proliferation and apoptosis as well as open field activity, with both ECs and neuronal cells exhibiting decreased proliferation, increased apoptosis, and pups exhibiting gender-specific differences in open field activities. Our studies demonstrate the autocrine and paracrine effects of EC HIF-1 α-modulating proliferative and apoptotic behaviors of EC and NPC in neurogenic regions of the brain and gender-specific behaviors in normoxic and hypoxic settings.

In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation. No increases in tyrosine phosphorylation of NEU1 or CD31 were detected. However, in postconfluent cells, we found increased tyrosine phosphorylation of a 120 kDa protein that was identified as p120 catenin (p120ctn). Prior silencing of c-src, fyn, or yes each reduced p120ctn phosphorylation. Prior knockdown of p120ctn prevented NEU1-CD31 association in both co-immunoprecipitation and pull-down assays. In these same assays, p120ctn associated with each of the four HPMEC-expressed SFKs as well as CD31 and NEU1. The CD31-p120ctn interaction was SFK-dependent whereas the NEU1-p120ctn interaction was not. Using purified recombinant binding partners in a cell-free system, direct protein-protein interactions between NEU1, CD31, and p120ctn were detected. Our combined data indicate that as HPMECs achieve confluence and CD31 ectodomains become homophilically engaged, multiple SFKs are activated to increase tyrosine phosphorylation of p120ctn, which in turn, functions as a cross-bridging adaptor molecule that physically couples NEU1 to CD31, permitting NEU1-mediated desialylation of CD31. These findings establish a SFK-driven, p120ctn-dependent mechanism for NEU1 recruitment to CD31.

Background The poor clinical results that are frequently reported for arteriovenous fistulae (AVF) for hemodialysis are typically due to failure of AVF maturation. We hypothesized that early AVF maturation is associated with generation of reactive oxygen species and activation of the hypoxia-inducible factor-1 (HIF-1) pathway, potentially promoting neointimal hyperplasia. We tested this hypothesis using a previously reported mouse AVF model that recapitulates human AVF maturation. Methods Aortocaval fistulae were created in C57Bl/6 mice and compared with sham-operated mice. AVFs or inferior vena cavas were analyzed using a microarray, Amplex Red for extracellular H2O2, quantitative polymerase chain reaction, immunohistochemistry, and immunoblotting for HIF-1α and immunofluorescence for NOX-2, nitrotyrosine, heme oxygenase-1 (HO-1), and vascular endothelial growth factor (VEGF)-A. Results Oxidative stress was higher in AVF than that in control veins, with more H2O2 (P = 0.007) and enhanced nitrotyrosine immunostaining (P = 0.005). Immunohistochemistry and immunoblot showed increased HIF-1α immunoreactivity in the AVF endothelium; HIF-1 targets NOX-2, HO-1 and VEGF-A were overexpressed in the AVF (P

Neutrophils from NOD (Non-Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes, and loss of directionality during chemotaxis (compared to wild-type [WT] C57BL/6 mice). Additionally, F-actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E

Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification., Generalized arterial calcification of infancy (GACI) is a terminal disease caused by the ENPP1 enzyme deficiency. Here, Albrigh et al. show that ENPP1 enzyme replacement therapy prevents the ectopic calcifications and mortality in mice with GACI, suggesting a novel treatment for vascular calcification in humans.

Background Atherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli. Materials and Methods TF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS). Results Mechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LF+Th for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PF+Th (14.8 ± 2.4) and TFF+Th (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th. Conclusions While uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.

Atherosclerosis is a chronic disease with systemic risk factors such as smoking, hypertension, hypercholesterolemia, diabetes, and male gender.1 Despite these risk factors, the localization of atherosclerosis is mainly observed in areas of complex arterial geometry, such as bifurcations, branching points, and curvatures.2 In these areas, the arterial wall receives complex physical forces generated by the pulsatile blood flow. The effects of hemodynamic forces in these areas have been postulated as a critical factor in atherogenesis.3 Numerous studies have focused on the response of endothelial cells (EC) to hemodynamic forces such as shear stress, pressure, and cyclic stretch and it is clear that these stimuli regulate protein synthesis, morphology, migration, apoptosis, proliferation, and survival in EC.4 Our laboratory has focused on the role of cyclic strain (CS), the repetitive, circumferential pulsatile pressure distention conferred to the vessel wall, and we have previously reported an increase in EC proliferation and expression of adhesion molecules, which can be involved in atherogenesis.5 The endothelium can be activated not only by mechanical forces but also by chemical stimuli, such as nicotine, lipopolysaccharide (LPS), tumor necrosis factor (TNF), and thrombin (Thr).6,7,8 Thr is a multifunctional serine protease that is not only involved in mediating the cleavage of fibrinogen to fibrin in the coagulation cascade but can also activate a variety of cell types, including platelets and EC. Thr signaling in the endothelium results in a multitude of phenotypic changes, including alterations in cell shape, permeability, vasomotor tone, leukocyte trafficking, migration, DNA synthesis, angiogenesis, and hemostasis.9 Thr has been demonstrated to increase the expression on EC of tissue factor (TF), a membrane-bound glycoprotein with a key role in the regulation of hemostasis.10 Under physiological conditions TF is virtually undetectable in EC but is present in abundant quantities in subendothelial cells such as smooth muscle cells and fibroblasts. Several studies have now shown a substantial increase in TF levels in advanced atherosclerotic lesions.11,12 For example, in the tissue factor pathway inhibitor (TFPI) knockout mice, significantly more atherosclerotic lesions were observed in areas of disturbed flow such as the carotid and iliac artery bifurcations, indicating that inhibition of TF activity by TFPI may be an important atheroprotective mechanism.13 Furthermore, analysis of atherosclerotic plaques has demonstrated TF in leukocytes, smooth muscle cells, and EC overlying the lesion.11 In vivo, EC are continuously exposed to mechanical forces and intermittently receive chemical stimuli. However, there are only few studies examining the effect of combined chemical and mechanical stimulation. The purpose of this study was to investigate the effect of CS, as a mechanical force, and Thr, as a chemical stress, on human umbilical vein endothelial cells (HUVEC) expression of TF and to elucidate the underlying mechanochemical signaling pathway.

Tissue factor (TF) is expressed in atherosclerotic lesions. Since mechanical forces influence endothelial cell (EC) function and are thought to account for the unique distribution of atherosclerosis in areas exposed to disturbed flow, we hypothesized that disturbed to-fro flow (TFF) and unidirectional pulsatile forward flow (PFF) would have different effects on TF expression in EC. TF RNA expression in HUVEC exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin was determined. TFF induced a significantly higher TF expression than PFF that was sustained for 8 h. Combination of mechanical and chemical stimuli induced significantly higher TF expression than only mechanical stresses, and this effect was synergistic in both TFF and PFF. The MAPK p38 inhibitor SB-203580 significantly inhibited TF expression induced by mechanical and chemical stimulations, but the MEK inhibitor PD-98059 did not inhibit TF induced by TFF. Immunoblotting revealed that ERK1/2 phosphorylation induced by TFF was sustained for 120 min, whereas that induced by PFF was not. We conclude that disturbed flow induced greater and sustained amplification of TF expression, and this synergistic effect may be regulated by p38 MAPK and ERK1/2. These results provide added insight into the mechanism of atherosclerosis in areas of disturbed flow.

There is little known about healing of the uterus after Cesarean delivery (CD). Uterine wound repair was studied by using two strains of mice with different wound healing characteristics: MRL/MpJ(+/+) (MRL: "high-healer" phenotype) and C57Bl/6 ("low-healer" phenotype). First, we examined the morphology and histology of the uterine wall repair. We identified wound granulation tissue 3 days post-CD in both strains, albeit less in the MRL strain. Macroscopically, no scar could be identified either in MRL or C57Bl/6 mice on day 60 post-CD. However, histologically, we found significant differences in wound integration, inflammation, and collagen birefringence between the two strains of mice. Using a histological index, we provided evidence for significant differences in mitotic activity in the initial phases of uterine healing among strains. Functional behavior of the uterine scar also was analyzed by using biomechanical parameters such as slope (measure of stiffness), yield point (measure of elasticity), and break point (measure of strength). There were significant differences in stiffness of the scarred myometrium between the two phenotypes. MRL mice displayed a significantly lower yield point compared with C57Bl/6. The break point was reached faster on days 15 and 60 in both C57Bl/6 and MRL strains compared with day 3 post-CD. Our findings indicate that differences in regenerative ability translate in histological, mitotic, and functional differences in biomechanical properties of the scarred myometrium after CD.

Premature infants have chronic hypoxia, resulting in cognitive and motor neurodevelopmental handicaps caused by suboptimal neural stem cell (NSC) repair/recovery in neurogenic zones (including the subventricular and the subgranular zones). Understanding the variable central nervous system repair response is crucial to identifying “at risk” infants and to increasing survival and clinical improvement of affected infants. Using mouse strains found to span the range of responsiveness to chronic hypoxia, we correlated differential NSC survival and self-renewal with differences in behavior. We found that C57BL/6 (C57) pups displayed increased hyperactivity after hypoxic insult; CD-1 NSCs exhibited increased hypoxia-induced factor 1α (HIF-1α) mRNA and protein, increased HIF-1α, and decreased prolyl hydroxylase domain 2 in nuclear fractions, which denotes increased transcription/translation and decreased degradation of HIF-1α. C57 NSCs exhibited blunted stromal-derived factor 1-induced migratory responsiveness, decreased matrix metalloproteinase-9 activity, and increased neuronal differentiation. Adult C57 mice exposed to hypoxia from P3 to P11 exhibited learning impairment and increased anxiety. These findings support the concept that behavioral differences between C57 and CD-1 mice are a consequence of differential responsiveness to hypoxic insult, leading to differences in HIF-1α signaling and resulting in lower NSC proliferative/migratory and higher apoptosis rates in C57 mice. Information gained from these studies will aid in design and effective use of preventive therapies in the very low birth weight infant population.

Particularities of the fetal immune response to infection cause a heightened inflammatory state that acts synergistically with microbial insult to induce damage. Proteomics offers the opportunity for detecting fetuses at risk of sepsis and neurological injury.Molecular tools (16S-rRNA) demonstrate that the diversity of microbial agents of intra-amniotic infection exceeds what is suspected clinically or is documented by cultures. The resulting inflammatory process has the potential to damage the fetus in utero. Stepwise algorithms (mass restricted score) have been developed to extract proteomic profiles characteristic of amniotic fluid inflammation. The mass restricted score includes four proteomic biomarkers: defensin-2, defensin-1, S100A12, and S100A8 proteins. Other amniotic fluid biomarkers relevant for preterm birth are S100A9 and insulin-like growth factor-binding protein 1. S100A12 - ligand for the receptor of advanced glycation end products - has the strongest association with histological chorioamnionitis and funisitis. Presence of S100A12 and S100A8 in amniotic fluid is predictive of early-onset neonatal sepsis and poor neurodevelopmental outcome.Presence of amniotic fluid proteomic biomarkers of inflammation is associated with increased inflammatory status of the fetus at birth. Future challenges are to find biomarkers that provide insight into molecular mechanisms of chronic fetal and neonatal cellular damage and to identify candidates for early neuroprotection strategies.

The lymph node vasculature is essential to immune function, but mechanisms regulating lymph node vascular maintenance and growth are not well understood. Vascular endothelial growth factor (VEGF) is an important mediator of lymph node endothelial cell proliferation in stimulated lymph nodes. It is expressed basally in lymph nodes and up-regulated upon lymph node stimulation, but the identity of VEGF-expressing cells in lymph nodes is not known. We show that, at homeostasis, fibroblast-type reticular stromal cells (FRC) in the T zone and medullary cords are the principal VEGF-expressing cells in lymph nodes and that VEGF plays a role in maintaining endothelial cell proliferation, although peripheral node addressin (PNAd)+ endothelial cells are less sensitive than PNAd− endothelial cells to VEGF blockade. Lymphotoxin β receptor (LTβR) blockade reduces homeostatic VEGF levels and endothelial cell proliferation, and LTβR stimulation of murine fibroblast-type cells up-regulates VEGF expression, suggesting that LTβR signals on FRC regulate lymph node VEGF levels and, thereby, lymph node endothelial cell proliferation. At the initiation of immune responses, FRC remain the principal VEGF mRNA-expressing cells in lymph nodes, suggesting that FRC may play an important role in regulating vascular growth in stimulated nodes. In stimulated nodes, VEGF regulates the proliferation and expansion of both PNAd+ and PNAd− endothelial cells. Taken together, these data suggest a role for FRC as paracrine regulators of lymph node endothelial cells and suggest that modulation of FRC VEGF expression may be a means to regulate lymph node vascularity and, potentially, immune function.

The αvβ3-integrin is expressed in angiogenic vessels in response to hypoxia and represents a potential novel target for imaging myocardial angiogenesis. This study evaluated the feasibility of noninvasively tracking hypoxia-induced αvβ3-integrin activation within the myocardium as a marker of angiogenesis early after myocardial infarction. Acute myocardial infarction was produced by coronary artery occlusion in rodent and canine studies. A novel 111In-labeled radiotracer targeted at the αvβ3-integrin (111In-RP748) was used to localize regions of hypoxia-induced angiogenesis early after infarction. In rodent studies, the specificity of 111In-RP748 for αvβ3-integrin was confirmed with a negative control compound (111In-RP790), and regional uptake of these compounds correlated with 201Tl perfusion and a 99mTc-labeled nitroimidazole (BRU59-21), which was used as a quantitative marker of myocardial hypoxia. The ex vivo analysis demonstrated that only 111In-RP748 was selectively retained in infarcted regions with reduced 201Tl perfusion and correlated with uptake of BRU59-21. In canine studies, myocardial uptake of 111In-RP748 was assessed using in vivo single-photon-emission computed tomography (SPECT), ex vivo planar imaging, and gamma well counting of myocardial tissue and correlated with 99mTc-labeled 2-methoxy-2-methyl-propyl-isonitrile (99mTc-sestamibi) perfusion. Dual-radiotracer in vivo SPECT imaging of 111In-RP748 and 99mTc-sestamibi provided visualization of 111In-RP748 uptake within the infarct region, which was confirmed by ex vivo planar imaging of excised myocardial slices. Myocardial 111In-RP748 retention was associated with histological evidence of αvβ3-integrin expression/activation in the infarct region. 111In-RP748 imaging provides a novel noninvasive approach for evaluation of hypoxia-induced αvβ3-integrin activation in myocardium early after infarction and may prove useful for directing and evaluating angiogenic therapies in patients with ischemic heart disease.

In vitro-formed bovine nucleus pulposus (NP) tissues were used as a model for tumor necrosis factor-alpha (TNF-alpha) induced NP degeneration.To elucidate the signal transduction mechanisms regulating TNF-alpha induced matrix metalloproteinase (MMP) activity.TNF-alpha is thought to contribute to the pathophysiology of intervertebral disc (IVD) degeneration by up-regulating MMPs, such as MMP-2. MMP-2 has been implicated in influencing disease progression and in the induction of neovascularization.In vitro-formed bovine NP tissues were treated with TNF-alpha to examine its effect on MMP-2 gene and protein levels and activity. The effect of TNF-alpha on membrane type (MT)1-MMP, an activator of MMP-2, was also assessed. MT1-MMP functional activation by TNF-alpha was confirmed using promoter-reporter luciferase constructs. Immunoblots and electrophoretic mobility shift assays were used to examine the expression and DNA binding activity of transcription factors known to regulate transcriptional activation of MT1-MMP.TNF-alpha treatment induced MMP-2 gelatinase activity, which occurred in the absence of any change in MMP-2 gene or protein expression, but did correlate with increased MT1-MMP mRNA and protein levels. Up-regulation of MMP-2 activity was dependent on the ERK-MAPK pathway. ERK-1/2 activation up-regulated early growth factor (Egr-1) expression and its DNA binding activity to the MT1-MMP promoter. There was no effect on Sp-1 binding activity. Reporter constructs demonstrated that TNF-alpha induced MT1-MMP transcriptional activation and that this response was dependant on ERK MAPK and Egr-1.TNF-alpha induced MMP-2 gelatinase activity correlated with induction of MT1-MMP and not MMP-2 expression. MMP-2 activation was dependent on the ERK-MAPK pathway. As ERK also appeared to regulate MT1-MMP production, this suggests that TNF-alpha induction of MMP-2 gelatinase activity may be regulated by MT1-MMP. These findings elucidate the regulation of gelatinase activity and identify a mechanism whereby TNF-alpha may contribute to matrix degradation in NP tissue.

Most tissues cannot survive without microvascular networks. In many cases, the host cannot vascularize implanted tissues, motivating the need for implantable vascular networks for tissue engineered grafts. However, engineering microvascular networks that are stable and functional for long times has proven challenging. The co-culture of neural progenitor cells with endothelial cells may lead to long term, functional microvascular networks. Ideally, these networks should be made from primary cells to avoid the potential safety concerns associated with immortalized or genetically-engineered cells. Thus, we have investigated and developed a paradigm for isolating and co-culturing primary rat endothelial cells and neural progenitor cells in biodegradable poly(ethylene glycol)/poly(L-lysine) macroporous hydrogels. The co-culture of these primary cells in the gels led to stabilization of vessels with no evidence of vessel regression even as far out as 6 weeks, the longest time point studied. Further more, the vessels contained host red blood cells, demonstrating they anastomosed with the host and were functional. Functional vessels were found throughout the implants, and no adverse effects such as clotting or thrombosis were observed. This work suggests that a co-culture of primary cells seeded in a macroporous hydrogel is a novel method to promote stable functional vascular networks which are critical for engineering complex tissues.

Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin–independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of αvβ3 integrin receptor and resulted in decreased αvβ3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT.

PECAM-1 (CD31) knockout (KO) mice exhibit excessive megakaryocytopoiesis accompanied by increased numbers of megakaryocytes associated with the stromal niche rather than the vascular niche. During earlier stages of megakaryocytopoiesis in KO marrow, an expanded Lin−Sca-1+ c-kit+ hematopoietic stem cell (HSC) population and increased quiescent Lin− progenitor pool were identified. During the later stages of megakaryocytopoiesis, CD31KO megakaryocytes exhibited abnormal adhesion/transmigration behaviors. Lastly, KO animals exhibited excessive splenic extramedullary megakaryocytopoiesis, which likely compensates for the impaired marrow megakaryocytopoiesis, resulting in normal peripheral platelet number. Thus, PECAM-1 modulates megakaryocytopoiesis in a hierarchic manner, functioning as a thermostat to “fine-tune” megakaryocytopoiesis.

In neutrophils, the major substrate of MAPKAPK2 (MK2) is an F-actin binding protein LSP1. Studies using mutants of the two potential Serine phosphorylation sites in LSP1 C-terminal F-actin binding region indicated that the major phosphorylation site for MK2 is Ser243 in murine neutrophils (Ser252 in humans). Human phosphoLSP1 antibodies that recognize phosphoSer252 site were prepared and revealed fMLP-induced neutrophil LSP1 phosphorylation. The phosphorylation was inhibited by p38 MAPK (upstream kinase for MK2) inhibitor SB203580. The antibodies also detect LSP1 phosphorylation in murine neutrophils. Immunostaining revealed that in WT murine neutrophils phosphoLSP1 was localized in F-actin enriched lamellipodia and oriented toward the fMLP gradient while non-phosphoLSP1 failed to colocalize with F-actin. In suspension, WT neutrophils exhibited persistent F-actin polarization following fMLP stimulation, while MK2(-/-) neutrophils exhibited transient F-actin polarization. These studies suggest that MK2-regulated LSP1 phosphorylation is involved in stabilization of F-actin polarization during neutrophil chemotaxis.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is known to inhibit platelet function and thrombus formation. The mechanisms involved in PECAM-1's roles as a modulator of hemostasis are still not completely understood. We examined the role of PECAM-1 as a regulator of tissue factor (TF) expression, a known important inducer of thrombosis. Wildtype and CD31KO mice underwent transient (30 min) renal ischemia followed by 24 h re-perfusion and their kidneys assessed for apoptosis, fibrin formation, and tissue factor expression. CD31KO mice exhibited increased tubular epithelial and endothelial apoptosis, increased fibrin deposition, and tissue factor expression. Human umbilical vein endothelial cells (HUVEC) transfected with antisense (AS) PECAM-1 oligonucleotides to downregulate PECAM-1 expression, exhibited greater induction of TF mRNA and protein expression as well as increased expression and nuclear localization of the transcription factor Egr-1 compared to scrambled AS PECAM-1 (Scr)-treated HUVEC following thrombin stimulation. TF induction was found to be mediated through thrombin receptor PAR-1 and the Galphai/o subunit of G-protein, confirmed by PAR-1 antagonist and pertussis toxin inhibition respectively. Thrombin-mediated TF induction was dependent on Rho Kinase activity, phosphorylation of p38(MAPK) and p85 & Akt dephosphorylation. The inverse correlation of PI3K-Akt phosphorylation with p38 (MAPK) phosphorylation was confirmed by pharmacological inhibition. These studies suggest that PECAM-1 is involved in regulating a signaling pathway, affecting PI3K and Akt activation, p38 (MAPK) phosphorylation, which in turn, affects Egr-1 expression and nuclear translocation, ultimately affecting TF expression. These findings provide new insights into the action of PECAM-1 as a modulator of thrombosis.

Juvenile hyaline fibromatosis (JHF) is a rare condition of childhood characterized by deposition of an amorphous substance of unclear nature in the dermis and subcutaneous tissues. The clinical picture includes painful skin lesions, leading to impairment of movements and severe disabilities. The allelic disease, infantile systemic hyalinosis (ISH), clinically overlaps with JHF but shows a worse picture with visceral involvement. Recently, germline mutations in the capillary morphogenesis gene-2 (CMG2) were found to be responsible for both diseases. Here, we present a case with classical clinicopathologic findings of JHF and features of ISH, and we describe a novel mutation in CMG2.

Premature infants are at an increased risk of developing cognitive and motor handicaps due to chronic hypoxia. Although the current therapies have reduced the incidence of these handicaps, untoward side effects abound. Using a murine model of sublethal hypoxia, we demonstrated reduction in several transcription factors that modulate expression of genes known to be involved in several neural functions. We demonstrate the induction of these genes by minocycline, a tetracycline antibiotic with noncanonical functions, in both in vitro and in vivo studies. Specifically, there was induction of genes, including Sox10 , Hif1a , Hif2a , Birc5 , Yap1 , Epo , Bdnf , Notch1 (cleaved), Pcna , Mag , Mobp , Plp1 , synapsin, Adgra2 , Pecam1 , and reduction in activation of caspase 3, all known to affect proliferation, apoptosis, synaptic transmission, and nerve transmission. Minocycline treatment of mouse pups reared under sublethal hypoxic conditions resulted in improvement in open field testing parameters. These studies demonstrate beneficial effects of minocycline treatment following hypoxic insult, document up-regulation of several genes associated with improved cognitive function, and support the possibility of minocycline as a potential therapeutic target in the treatment of neurodevelopmental handicaps observed in the very premature newborn population. Additionally, these studies may aid in further interpretation of the effects of minocycline in the treatment trials and animal model studies of fragile X syndrome and multiple sclerosis.

Laminar shear stress (SS) induces an antiproliferative and anti-inflammatory endothelial phenotype and increases Klf2 expression. We altered the diameter of an arteriovenous fistula (AVF) in the mouse model to determine whether increased fistula diameter produces disturbed SS in vivo and if acutely increased disturbed SS results in decreased Klf2 expression. The mouse aortocaval fistula model was performed with 22, 25, or 28 gauge needles to puncture the aorta and the inferior vena cava. Duplex ultrasound was used to examine the AVF and its arterial inflow and venous outflow, and SS was calculated. Arterial samples were examined with western blot, immunohistochemistry, and immunofluorescence analysis for proteins and qPCR for RNA. Mice with larger diameter fistulae had diminished survival but increased AVF patency. Increased SS magnitudes and range of frequencies were directly proportional to the needle diameter in the arterial limb proximal to the fistula but not in the venous limb distal to the fistula, with 22-gauge needles producing the most disturbed SS in vivo. Klf2 mRNA and protein expression was diminished in the artery proximal to the fistula in proportion to increasing SS. Increased fistula diameter produces increased SS magnitude and frequency, consistent with disturbed SS in vivo. Disturbed SS is associated with decreased mRNA and protein expression of Klf2. Disturbed SS and reduced Klf2 expression near the fistula are potential therapeutic targets to improve AVF maturation.

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and γδ T cells are involved in the protective immune response against viral challenge. We have now examined whether γδ T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRδ−/− mice survived primary infection with WN virus compared with 80–85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of γδ T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, γδ T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8+ T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8+ T cells from TCRδ−/− mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that γδ T cells directly link innate and adaptive immunity during WN virus infection.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates a variety of endothelial and immune cell biological responses. PECAM-1-null mice exhibit prolonged and increased permeability after inflammatory insults. We observed that in PECAM-1-null endothelial cells (ECs), beta-catenin remained tyrosine phosphorylated, coinciding with a sustained increase in permeability. Src homology 2 domain containing phosphatase 2 (SHP-2) association with beta-catenin was diminished in PECAM-1-null ECs, suggesting that lack of PECAM-1 inhibits the ability of this adherens junction component to become dephosphorylated, promoting a sustained increase in permeability. beta-Catenin/Glycogen synthase kinase 3 (GSK-3beta) association and beta-catenin serine phosphorylation levels were increased and beta-catenin expression levels were reduced in PECAM-1-null ECs. Glycogen synthase kinase 3 (GSK-3beta) serine phosphorylation (inactivation) was blunted in PECAM-1-null ECs after histamine treatment or shear stress. Our data suggest that PECAM-1 serves as a critical dynamic regulator of endothelial barrier permeability. On stimulation by a vasoactive substance or shear stress, PECAM-1 became tyrosine phosphorylated, enabling recruitment of SHP-2 and tyrosine-phosphorylated beta-catenin to its cytoplasmic domain, facilitating dephosphorylation of beta-catenin, and allowing reconstitution of adherens junctions. In addition, PECAM-1 modulated the levels of beta-catenin by regulating the activity of GSK-3beta, which in turn affected the serine phosphorylation of beta-catenin and its proteosomal degradation, affecting the ability of the cell to reform adherens junctions in a timely fashion.

Objective To test the effects of a novel tylophorine analog, DCB 3503, on the prevention and treatment of collagen-induced arthritis (CIA) and to elucidate its underlying mechanisms. Methods DBA/1J mice were immunized with type II collagen, and in some cases, lipopolysaccharide (LPS) was used to boost the development of arthritis. DCB 3503 was injected intraperitoneally before or after the onset of CIA. Mice were monitored to assess the effects of DCB 3503 on the clinical severity of the disease, and pathologic changes in the joints were examined histologically. Levels of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in serum and joint tissues were measured by enzyme-linked immunosorbent assay and by cytometric bead array analysis. The effect of DCB 3503 on LPS-induced proinflammatory cytokines from bone marrow–derived dendritic cells was determined by flow cytometry. Results DCB 3503 significantly suppressed the development and progression of CIA. Moreover, DCB 3503 completely blocked the LPS-triggered acceleration of joint inflammation and destruction. Consistent with its effects in vivo, DCB 3503 significantly suppressed the synthesis of proinflammatory cytokines in inflamed joints as well as cytokine synthesis by macrophages examined ex vivo. Treatment also reduced the levels of inflammatory cytokines (IL-6, IL-12, TNFα, and monocyte chemotactic protein 1) produced by bone marrow–derived dendritic cells in vitro. However, DCB 3503 showed no direct effects on T cell proliferation and B cell antibody response. Conclusion Because of its ability to specifically suppress innate immune responses, DCB 3503 may be a novel therapeutic agent for inflammatory arthritis in humans.

Wobbly Hedgehog Syndrome (WHS) is a progressive paralysis that occurs in approximately 10% of pet African hedgehogs in North America. Clinical signs of WHS begin with mild ataxia, progress to more severe neurologic signs, and ultimately lead to complete paralysis. The onset of WHS commonly occurs under 2 years of age, but can occur at any age. Progression rate is variable, and the majority of hedgehogs are completely paralyzed by 15 months after the onset of clinical signs. WHS can only be definitively diagnosed by post-mortem examination of tissues from the central nervous system. The characteristic histopathology of WHS is vacuolization of the white matter of the brain and spinal cord, and associated neurogenic muscle atrophy. There is no inflammation of the central nervous system associated with WHS. The etiology of WHS is unknown, but pedigree analysis indicates a familial tendency to the disease.

Background— Noninvasive imaging strategies play a critical role in assessment of the efficacy of angiogenesis therapies. The α v β 3 integrin is activated in angiogenic vessels and represents a potential target for noninvasive imaging of angiogenesis. Methods and Results— We evaluated a 99m Tc-labeled peptide (NC100692) targeted at α v β 3 integrin for imaging in an established murine model of angiogenesis induced by hindlimb ischemia. Control mice (n=9) or mice with surgical right femoral artery occlusion (n=29) were injected with NC100692 (1.5±0.2 mCi IV) at different times after femoral occlusion (1, 3, 7, and 14 days) for in vivo pinhole planar gamma camera imaging. Tissue from hindlimb proximal and distal to occlusion was excised for gamma well counting and for immunostaining. On in vivo pinhole images, increased focal NC100692 activity was seen distal to the occlusion at days 3 and 7. This increase in relative NC100692 activity was confirmed by gamma well counting. Lectin staining confirmed increased angiogenesis in the ischemic hindlimb at these time points. A fluorescent analogue of NC100692 was used to confirm specificity and localization of the targeted tracer in cultured endothelial cells. In addition, endothelial cell specificity was confirmed on tissue sections with the use of dual immunofluorescent staining of endothelium and the fluorescent analogue targeted at the α v β 3 integrin. Conclusions— A 99m Tc-labeled peptide (NC100692) targeted at α v β 3 integrin selectively localized to endothelial cells in regions of increased angiogenesis and could be used for noninvasive serial “hot spot” imaging of angiogenesis. This targeted radiotracer imaging approach is a major advance in tracking therapeutic myocardial angiogenesis and has an important clinical potential.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated beta-catenin and modulates beta-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated beta-catenin, and to a lesser extent unphosphorylated beta-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for beta-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, gamma-catenin-PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1-gamma-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S(673) to C and D and S(669 and 670) to C, we confirmed the importance of S(673) and its phosphorylation state as a mediator of gamma-catenin-PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1-catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions.

PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK½, p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), an adhesion molecule expressed on hematopoietic and endothelial cells, mediates apoptosis, cell proliferation, and migration and maintains endothelial integrity in addition to its roles as a modulator of lymphocyte and platelet signaling and facilitator of neutrophil transmigration. Recent data suggest that CD31 functions as a scaffolding protein to regulate phosphorylation of the signal transducers and activators of transcription (STAT) family of signaling molecules, particularly STAT3 and STAT5. STAT3 regulates the acute phase response to innate immune stimuli such as lipopolysaccharide (LPS) and promotes recovery from LPS-induced septic shock. Here we demonstrate that CD31-deficient mice have reduced survival during endotoxic LPS-induced shock. As compared to wild-type controls, CD31-deficient mice showed enhanced vascular permeability; increased apoptotic cell death in liver, kidney, and spleen; and elevated levels of serum tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFNgamma), MCP-1, MCP-5, sTNRF, and IL-6. In response to LPS in vivo and in vitro, splenocytes and endothelial cells from knockout mice had reduced levels of phosphorylated STAT3. These results suggest that CD31 is necessary for maintenance of endothelial integrity and prevention of apoptosis during septic shock and for STAT3-mediated acute phase responses that promote survival during septic shock.

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2)hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.