Endothelial Cell Phenotype Research Articles

Type I interferon activation and endothelial dysfunction in caveolin-1 insufficiency-associated pulmonary arterial hypertension

Interferonopathies, interferon (IFN)-α/β therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.

Small Chain Fatty Acid Phenylbutyric Acid Alleviated Inflammation-Induced Endoplasmic Reticulum Stress in Endothelial Cells

Endothelial cells (EC) have dynamic properties and high plasticity in response to microenvironmental change. A proinflammatory cytokine such as tumor necrotizing factor-α (TNF-α) can induce EC phenotype shift to osteoinduction properties by releasing a potent osteogenic cytokine, namely bone morphogenetic protein 2 (BMP2). Normally BMP2 acts as an osteoblast stimulating factor in bone and cartilage tissue. BMP2 activation in vascular tissue will invite osteoblast recruitment and mineralization and generated pathological vascular stiffening and calcification. Recently, endoplasmic reticulum stress (ERS) has been emerging as a new target therapy in many vascular diseases such as vascular stiffening and calcification. Some short-chain fatty acid like 4-phenyl butyric acid has been shown had anti-ERS properties. However, the role of 4-phenyl butyric acid in BMP2 inhibition in endothelial cells is still poorly understood. Hence, we investigated the role of 4-phenyl butyric acid in inflammation-induced BMP2 expression in human vein derived endothelial cells. Endothelial cells obtained from a baby born umbilical vein were cultured and pre-treated with TNF-α (5 ng/ml) as inflammation precondition. Multiple doses of 4-phenyl butyrate acid (4- PBA) 1 nM/mL, 2 nM/mL, and 3 nM/m were used as ERS inhibitors. The expression of two ERS biomarkers, glucose-related protein-8 (GRP78) and activating transcription factor-6 (ATF6), were measured. Statistical analysis was done using one-way ANOVA and Kruskal Wallis tests, and P<0.01 considered as significant. 4- PBA decrease luminal BMP2 at dose one nM/L, GRP78 at dose 1 nM/L, and translocated ATF6 expression at dose 1 nM/L in endothelial culture dose-dependently. Short-chain fatty acid 4-phenylbutyrate acid decreases luminal ERS marker GRP78 and translocated ATF6 expression in endothelial culture. ERS has a role in osteoinductive phenotype shifting in inflammation endothelial cells, which was the novelty of this research. Further research needs to elucidate ERS inhibition in in vivo experiment.

Enhanced proliferation and angiogenic phenotype of endothelial cells via negatively-charged alginate and chondroitin sulfate microsphere hydrogels

Sodium alginate-based hydrogel was the one of the most used polymers for cell delivery. However, the adsorption of extracellular matrix and proteins was inhibited due to the formation of a hydrated surface layer of these hydrogels. In this study, a novel cell delivery system, negatively-charged alginate and chondroitin sulfate microsphere hydrogel (nCACSMH), was fabricated with excellent permeability and biocompatibility in the action of a high voltage direct-current electric field. Negative charge was introduced to the surface of nCACSMH to obtain the expanded network and enhanced permeability. Additionally, the increasing content of chondroitin sulfate in nCACSMH could give rise to the charge density and its asymmetric structure, thus the uneven, plicate and expanded surface of nCACSMH which was favorable to cell proliferation was developed. Moreover, chondroitin sulfate was released with the degradation of nCACSMH, which played a crucial role in maintaining the normal physiological functions of cells. Thus the proliferation of human umbilical vein endothelial cells (HUVECs) was further accelerated and the angiogenesis related genes expression in endothelial cells was continuously and dramatically up-regulated. After 4 d, the proliferation and viability of HUVECs were significantly improved, the cells were distributed evenly in nCACSMH. The novel nCACSMH has the potential to be used as cell delivery, three-dimensional (3D) cell cultures for cell therapy, 3D bioprinting, high-throughput screening for drugs, and disease model for regeneration and constructing of tissue engineering.

Early events in endothelial flow sensing.

Responses of vascular and lymphatic endothelial cells (ECs) to fluid shear stress (FSS) from blood or lymphatic fluid flow govern the development, physiology, and diseases of these structures. Extensive research has characterized the signaling, gene expression and cytoskeletal pathways that mediate effects on EC phenotype and vascular morphogenesis. But the primary mechanisms by which ECs transduce the weak forces from flow into biochemical signals are less well understood. This review covers recent advances in our understanding of the immediate mechanisms of FSS mechanotransduction, integrating results from different disciplines, addressing their roles in development, physiology and disease, and suggesting important questions for future work.

Tissue-specific endothelial cell heterogeneity contributes to unequal inflammatory responses

Endothelial cells (EC) coordinate vascular homeostasis and inflammation. In organ transplantation, EC are a direct alloimmune target. We posited that tissue specific heterogeneity of vascular EC may partly underlie the disparate organ-specific alloimmune risk. We examined the vascular endothelial response to inflammation across six primary endothelial beds from four major transplanted organs: the heart, lung, kidney and liver. First, we reanalyzed a public dataset of cardiac allograft rejection and found that endothelial inflammatory response genes were elevated in human cardiac allograft biopsies undergoing rejection compared with stable grafts. Next, the inducible inflammatory phenotypes of EC from heart, lung, kidney, and liver were characterized in vitro, focused on expression of adhesion molecules and chemokines, and recruitment of allogeneic peripheral blood mononuclear immune cells. Large vessel cardiac EC most highly upregulated VCAM-1, particularly compared with hepatic EC, supported greater leukocyte adhesion and had distinct chemokine profiles after stimulation with cytokines and complement. Differentially expressed gene candidates that are known regulators of cytokine signaling and inflammatory programming were verified in publicly available datasets of organ-specific endothelial transcriptomes. In summary, differential baseline expression of immune regulating genes may contribute to differential vascular inflammatory responses depending on organ.

Presence of a mutation in PSEN1 or PSEN2 gene is associated with an impaired brain endothelial cell phenotype in vitro

BackgroundAlzheimer’s disease (AD) is the most common form of neurodegenerative disease. It is an irreversible condition marked by irreversible cognitive loss, commonly attributed to the loss of hippocampal neurons due to the formation of senile plaques and neurofibrillary tangles. Although the sporadic form is the most prevalent, the presence of familial form (involving several genes such as APP, PSEN1, and PSEN2) of the disease is commonly used as a model for understanding the pathophysiology of the disease. The aim of this study is to investigate the effect of a mutation on PSEN1 and PSEN2 genes on the BBB function using induced pluripotent stem cells (iPSCs).Methods iPSC lines from patients suffering from a familial form of Alzheimer’s disease and harboring mutations in PSEN1 or PSEN2 were used in this study and compared to a control iPSC line. Cells were differentiated into brain microvascular endothelial cells (BMECs) following established differentiation protocols. Barrier function was assessed by measuring TEER and fluorescein permeability, drug transporter activity was assessed by uptake assay, glucose uptake and metabolism assessed by cell flux analyzer, mitochondrial potential by JC-1, and lysosomal acidification by acridine orange.ResultsiPSC-derived BMECs from the FAD patient presenting a mutation in the PSEN1 gene showed impaired barrier function compared to the FAD patient harboring a mutation in PSEN2 and to the control group. Such impaired barrier function correlated with poor tight junction complexes and reduced drug efflux pump activity. In addition, both PSEN1 and PSEN2-BMECs displayed reduced bioenergetics, lysosomal acidification, autophagy, while showing an increase in radical oxygen species (ROS) production. Finally, PSEN1- and PSEN2-BMECs showed an elevated secretion of Aβ1–40 peptides compared to control-BMECs.ConclusionsOur study reports that iPSC-derived BMECs obtained from FAD patients showed impaired barrier properties and BMEC metabolism. In particular, mutation in the PSEN1 gene was associated with a more detrimental phenotype than mutation in PSEN2, as noted by a reduced barrier function, reduced drug efflux pump activity, and diminished glucose metabolism. Therefore, assessing the contribution of genetic mutations associated with Alzheimer’s disease will allow us to better understand the contribution of the BBB in dementia, but also other neurodegenerative diseases.

Ethanol Gestational Exposure Impairs Vascular Development and Endothelial Potential to Control BBB-Associated Astrocyte Function in the Developing Cerebral Cortex.

Ethanol consumption during pregnancy or lactation period can induce permanent damage to the development of the central nervous system (CNS), resulting in fetal alcohol spectrum disorders (FASD). CNS development depends on proper neural cells and blood vessel (BV) development and blood-brain barrier (BBB) establishment; however, little is known about how ethanol affects these events. Here, we investigated the impact of ethanol exposure to endothelial cells (ECs) function and to ECs interaction with astrocytes in the context of BBB establishment. Cerebral cortex of newborn mice exposed in utero to ethanol (FASD model) presented a hypervascularized phenotype, revealed by augmented vessel density, length, and branch points. Further, aberrant distribution of the tight junction ZO-1 protein along BVs and increased rates of perivascular astrocytic endfeet around BVs were observed. In vitro exposure of human brain microcapillary ECs (HBMEC) to ethanol significantly disrupted ZO-1 distribution, decreased Claudin-5 and GLUT-1 expression and impaired glucose uptake, and increased nitric oxide secretion. These events were accompanied by upregulation of angiogenesis-related secreted proteins by ECs in response to ethanol exposure. Treatment of cortical astrocytes with conditioned medium (CM) from ethanol exposed ECs, upregulated astrocyte's expression of GFAP, Cx43, and Lipocalin-2 genes, as well as the pro-inflammatory genes, IL-1beta, IL-6, and TNF-alpha, which was accompanied by NF-kappa B protein nuclear accumulation. Our findings suggest that ethanol triggers a dysfunctional phenotype in brain ECs, leading to impairment of cortical vascular network formation, and promotes ECs-induced astrocyte dysfunction, which could dramatically affect BBB establishment in the developing brain.

Microvesicles produced by monocytes affect the phenotype and functions of endothelial cells

<abstract> Monocytes\macrophages regulate angiogenesis via cytokine production and contact interactions with endothelial cells (ECs). The biological effects of macrophage-derived microvesicles (MVs) are studied using cell lines, such as monocytic leukemia THP-1 cell line. The effect of MVs produced by THP-1 cells on EC phenotype and functions remain understudied. In this research, we studied the effect of MVs produced by THP-1 cells on the phenotype, proliferation, migration, and vascular formation of EA.Hy926 ECs. MVs produced by THP-1 cells express CD54, CD18, CD11a, CD11b, CD29, CD120a, CD120b, VEGFR1, VEGFR2, CD105, CD119, TGFR2 on the surface and contain ERK1/2, pERK1/2 Akt, FGF10, endothelin-2. The transfer of an intracellular protein labeled with a fluorescent dye from MVs produced by THP-1 cells to EA.Hy926 ECs was established. It was found that MVs derived from THP-1 cells inhibit EC proliferation. In high concentrations, MVs reduce EC migration, increase the length but decrease the number of vessels formed by ECs, promoting the development of non-branching angiogenesis. On the contrary, in low concentrations, MVs increase EC migration, reduce the length, and increase the number of vessels formed by ECs, promoting the development of branching angiogenesis. Thus, the fundamental possibility of the influence of MVs produced by THP-1 cells on the processes of angiogenesis has been established. Proteins found in the MVs composition may be responsible for the observed effects of MVs on ECs. </abstract>

Aberrant expression of proatherogenic cytokines and growth factors in human umbilical vein endothelial cells from newborns of type 2 diabetic women

Objectives:This study reports the levels of cytokines, chemokines, and growth factors previously identified as taking part in the pathology of atherosclerosis in human umbilical vein endothelial cells derived from mothers with type 2 diabetes and compares them with those in human umbilical vein endothelial cells derived from healthy mothers under normal glucose conditions.Methods:Cytokine analysis measures of human umbilical vein endothelial cell lysates were obtained using a multiple analyte profiling (xMAP) assay based on magnetic bead-based technology, using the MAGPIX instrument. The correlation between cytokines, chemokines, and growth factors was examined statistically in human umbilical vein endothelial cells derived from mothers with type 2 diabetes.Results:This study showed that the expression of proinflammatory cytokine interleukin-1 alpha was significantly greater in human umbilical vein endothelial cells derived from mothers with type 2 diabetes than those derived from healthy mothers. The protein level of granulocyte colony-stimulating factor was higher in human umbilical vein endothelial cells derived from mothers with type 2 diabetes than those derived from healthy mothers. A significant positive correlation was demonstrated between the protein expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in human umbilical vein endothelial cells derived from mothers with type 2 diabetes.Conclusion:Diabetes evokes a persistent inflammatory phenotype in human umbilical vein endothelial cells, as indicated by the enhanced production of cytokines and growth factors under normal glucose conditions.

Pan-PPAR agonist lanifibranor improves portal hypertension and hepatic fibrosis in experimental advanced chronic liver disease

In advanced chronic liver disease (ACLD), deregulated hepatic necroinflammatory processes play a key role in the development of liver microvascular dysfunction, fibrogenesis, and increased hepatic vascular tone, resulting in progression of ACLD and portal hypertension. Given the current lack of an effective treatment, we aimed to characterise the effects of the pan-peroxisome proliferator-activated receptor (pan-PPAR) agonist lanifibranor in 2 preclinical models of ACLD, as well as in liver cells from patients with ACLD. Cirrhotic rats (thioacetamide or common bile duct ligation; TAA or cBDL) randomly received lanifibranor (100 mg/kg/day, po) or vehicle for 14 days (n= 12/group). PPAR expression, systemic and hepatic haemodynamics, presence of ascites, liver sinusoidal endothelial cell (LSEC) phenotype, hepatic stellate cell (HSC) activation, serum transaminases and albumin, hepatic macrophage infiltration, cytokine expression, and liver fibrosis were determined. Hepatic cells were isolated from the livers of patients with cirrhosis and their phenotype was evaluated after treatment with either lanifibranor or vehicle. TAA-cirrhotic rats receiving lanifibranor showed significantly lower portal pressure compared with vehicle-treated animals (-15%; p = 0.003) without decreasing portal blood flow, indicating improved hepatic vascular resistance. Moreover, lanifibranor-treated TAA-rats showed decreased ascites, improved LSEC and HSC phenotypes, ameliorated hepatic microvascular function, reduced hepatic inflammation, and significant fibrosis regression (-32%; p = 0.020). These findings were confirmed in the cBDL rat model as well as in human liver cells from patients with cirrhosis, which exhibited phenotypic improvement upon treatment with lanifibranor. Lanifibranor ameliorates fibrosis and portal hypertension in preclinical models of decompensated cirrhosis. Promising results in human hepatic cells further support its clinical evaluation for the treatment of ACLD. Advanced chronic liver disease (ACLD) constitutes a serious public health issue for which safe and effective treatments are lacking. This study shows that lanifibranor improves portal hypertension and liver fibrosis, 2 key elements of the pathophysiology of ACLD, in preclinical models of the disease. Evaluation of lanifibranor in liver cells from patients with ACLD further supports its beneficial effects.

Endothelial JAK2V617F mutation leads to thrombosis, vasculopathy, and cardiomyopathy in a murine model of myeloproliferative neoplasm

ObjectiveCardiovascular complications are the leading cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). The acquired kinase mutation JAK2V617F plays a central role in these disorders. Mechanisms responsible for cardiovascular dysfunction in MPNs are not fully understood, limiting the effectiveness of current treatment. Vascular endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in patients with MPNs. The goal of this study was to test the hypothesis that the JAK2V617F mutation alters endothelial function to promote cardiovascular complications in patients with MPNs. Approach and ResultsWe employed murine models of MPN in which the JAK2V617F mutation is expressed in specific cell lineages. When JAK2V617F is expressed in both blood cells and vascular ECs, the mice developed MPN and spontaneous, age‐related dilated cardiomyopathy with an increased risk of sudden death as well as a prothrombotic and vasculopathy phenotype on histology evaluation. In contrast, despite having significantly higher leukocyte and platelet counts than controls, mice with JAK2V617F‐mutant blood cells alone did not demonstrate any cardiac dysfunction, suggesting that JAK2V617F‐mutant ECs are required for this cardiovascular disease phenotype. Furthermore, we demonstrated that the JAK2V617F mutation promotes a pro‐adhesive, pro‐inflammatory, and vasculopathy EC phenotype, and mutant ECs respond to flow shear differently than wild‐type ECs. ConclusionsThese findings suggest that the JAK2V617F mutation can alter vascular endothelial function to promote cardiovascular complications in MPNs. Therefore, targeting the MPN vasculature represents a promising new therapeutic strategy for patients with MPNs.

Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells

Activation of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is critical for vascular endothelial redox homeostasis in regions of high, unidirectional shear stress (USS), however the underlying mechanosensitive mediators are not fully understood. The endothelial glycocalyx is disrupted in arterial areas exposed to disturbed blood flow that also exhibit enhanced oxidative stress leading to atherogenesis. We investigated the contribution of glycocalyx sialic acids (SIA) to Nrf2 signaling in human endothelial cells (EC) exposed to atheroprotective USS or atherogenic low oscillatory shear stress (OSS). Cells exposed to USS exhibited a thicker glycocalyx and enhanced turnover of SIA which was reduced in cells cultured under OSS. Physiological USS, but not disturbed OSS, enhanced Nrf2-mediated expression of antioxidant enzymes, which was attenuated following SIA cleavage with exogenous neuraminidase. SIA removal disrupted kinase signaling involved in the nuclear accumulation of Nrf2 elicited by USS and promoted mitochondrial reactive oxygen species accumulation. Notably, knockdown of the endogenous sialidase NEU1 potentiated Nrf2 target gene expression, directly implicating SIA in regulation of Nrf2 signaling by USS. In the absence of SIA, deficits in Nrf2 responses to physiological flow were also associated with a pro-inflammatory EC phenotype. This study demonstrates that the glycocalyx modulates endothelial redox state in response to shear stress and provides the first evidence of an atheroprotective synergism between SIA and Nrf2 antioxidant signaling. The endothelial glycocalyx therefore represents a potential therapeutic target against EC dysfunction in cardiovascular disease and redox dyshomeostasis in ageing.

The Relationship Between Reactive Oxygen Species and Endothelial Cell Metabolism.

Cardiovascular disease (CVD) has been the leading cause of death for many decades, highlighting the importance of new research and treatments in the field. The role of hypoxia and subsequent free radical production [reactive oxygen species (ROS)] have become an area of particular interest in CVD. Interestingly, our laboratory and other laboratories have recently reported positive roles of subcellular ROS in modulating endothelial cell (EC) metabolism, proliferation, and angiogenesis. This bidirectional relationship between ROS and EC metabolism, as well as functional changes, continues to be an area of active research. Interestingly, ECs have been shown to rely on anaerobic processes for ATP generation, despite their direct access to oxygen. This paradox has proven to be beneficial as the major reliance on glycolysis produces ATP faster, preserves oxygen, and results in reduced ROS levels in contrast to oxidative phosphorylation. This review will address the relationship between ROS and carbohydrate, lipid, and nitrogen metabolism in ECs, and their effects on EC phenotype such as sprouting angiogenesis.

Abstract 15624: The Anti-lung Cancer Drug (R)-crizotinib Predisposes and Exacerbates Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent elevation of pulmonary vascular resistance leading to right heart failure and death. In 10% of cases, PAH is induced by drugs including chemotherapeutic agents such as RTK inhibitors (e.g. dasatinib). (R)-Crizotinib is an ALK/ROS1/MET inhibitor increasingly used for the treatment of non-small cell lung cancer (NSCLC). Interestingly, (R)-Crizotinib has been shown to induce endothelial cells (EC) dysfunction and is associated with dyspnea and peripheral oedema, which are central symptoms of PAH. We thus hypothesized that chronic administration of (R)-Crizotinib exacerbates and predisposes PAH. Material and results: We found that daily administration of (R)-Crizotinib (100mg/kg for 2 weeks) in Sugen/Hypoxia (Su/Hx) PAH rat model significantly increased mortality rates. Compared to vehicle-treated rats, (R)-Crizotinib was associated with worsening of hemodynamic parameters (RVSP, mPAP, SV, CO and TPR measured by right heart catheterization; p&lt;0.05; n=4-6). Histologically, (R)-Crizotinib enhanced pulmonary arteries (PA) wall thickness (EVG stain; p&lt;0.05) and increased fibrosis and macrophages accumulation in the lungs and right ventricle. In addition, we showed that pretreatment of rats with (R)-Crizotinib exaggerates the response to monocrotaline (MCT, 40mg/kg), as revealed by histological (increase in vascular remodeling) and hemodynamic measurements (mPAP; TPR; CO) (p&lt;0.01; n=7-10). In vitro, (R)-Crizotinib reduced MET and AKT activation (WB) in human PA endothelial cells promoting endothelial dysfunction via decreased proliferation (Ki67) and increased apoptosis (Annexin V) rates. In addition, (R)-Crizotinib interferes with autophagy flux (increase in LC3-II and p62 accumulation) and was accompanied by the appearance of atypical morphological phenotypes in PA endothelial cells (multinucleated cells). Conclusion: We showed for the first time that (R)-Crizotinib treatment predisposes and exacerbates PAH in animal models (Sugen/Hypoxia and monocrotaline). Understanding the mechanism will provide promising perspectives for a better management of patients treated with (R)-Crizotinib or ALK/ROS1/MET inhibitors.

Abstract 14925: Mechanical Tension in Syndecan-1 is Regulated by Extracellular Mechanical Cues and Fluidic Shear Stress

Syndecan-1 (SDC1) is a transmembrane proteoglycan that mediates the shear stress-induced signaling and inflammatory phenotypes of endothelial cells. While SDC1 is known to be involved mechanically in regulating the behavior of cells, it remains unknown how SDC1 responds to extracellular mechanical cues on the molecular level. We designed a set of FRET-based SDC1 tension sensors including full-length SDC1 (SDC1TS), SDC1 with deleted ectodomain (ΔEcto), SDC1 with deleted glycosylation sites (ΔGAG) and SDC1 with a deleted cytoplasmic tail (ΔCyto). We transduced these constructs into ECs and validated the constructs by WB and confocal imaging. When cultured on glass-bottom coverslips, we found that the baseline tension in SDC1TS was significantly higher than that in Ecto and GAG mutants. When cultured on engineered substrates, we demonstrated that tension in SDC1 is modulated by micropatterned surfaces and nanotopographical cues. To study if SDC1 directly responds to substrate stiffness, transduced ECs were cultured on 0.2 kPa and 25 kPa substrates. We found that there was significantly decreased tension in the Ecto construct compared to the full-length SDC1TS construct on 0.2 kPa substrates but no significant changes were observed on the 25 kPa substrates. To examine the effect of substrate stiffness on the association of SDC1 with adhesion-related proteins, we performed IP and WB on ECs expressing HA-tagged SDC1 cultured on 0.2 kPa or 25 kPa substrates. We found there were increases in SDC1 binding to action and myosin IIb, while there were decreased in SDC1 binding to Src, PKA, and FAK in cells grown on stiff substrates versus on soft substrate. To study the effect of shear stress on the tension of SDC1, ECs expressing the constructs were cultured on static or under 12dyn/cm 2 fluidic shear stress. We found that a gradient developed in the tension of SDC1 in all four constructs. There was higher tension of SDC1 in the upstream region in comparison to the downstream region. IP and WB results showed that shear stress-induced SDC1 association with actin, Src, myosin IIb, cortactin, calmodulin, and integrin beta3. In summary, our results demonstrate that SDC1 is mechanically responsive to substrate mediated biophysical cues and shear stress.

The six-transmembrane protein Stamp2 ameliorates pulmonary vascular remodeling and pulmonary hypertension in mice

Six-transmembrane protein of prostate (Stamp2) protects from diabetes and atherosclerosis in mice via anti-inflammatory mechanisms. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PAH), we investigated the role of Stamp2. Stamp2 expression was substantially reduced in the lung of humans with idiopathic PAH, as well as in experimental PAH. In Stamp2-deficient mice, hypoxia modestly aggravated pulmonary vascular remodeling and right ventricular pressure compared to WT. As endothelial cell (EC) and pulmonary arterial smooth muscle cell (PASMC) phenotypes drive remodeling in PAH, we explored the role of Stamp2. Knock-down of Stamp2 in human EC neither affected apoptosis, viability, nor release of IL-6. Moreover, Stamp2 deficiency in primary PASMC did not alter mitogenic or migratory properties. As Stamp2 deficiency augmented expression of inflammatory cytokines and numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages may drive vascular remodeling. Thus, PASMC responses were assessed following treatment with conditioned media of primary Stamp2−/− or WT macrophages. Stamp2−/− supernatants induced PASMC proliferation and migration stronger compared to WT. A cytokine array revealed CXCL12, MCP-1 and IL-6 as most relevant candidates. Experiments with neutralizing antibodies confirmed the role of these cytokines in driving Stamp2’s responses. In conclusion, Stamp2 deficiency aggravates pulmonary vascular remodeling via cross-talk between macrophages and PASMC. Despite a substantial pro-inflammatory response, the hemodynamic effect of Stamp2 deficiency is modest suggesting that additional mechanisms apart from inflammation are necessary to induce severe PAH.

Adaptation of endothelial cells to shear stress under atheroprone conditions by modulating internalization of vascular endothelial cadherin and vinculin.

BackgroundEndothelial cells play a pivotal role in cardiovascular physiology and pathology by providing a barrier to the bloodstream. In the current study, we investigated the phenotype and barrier function of endothelial cells in response to shear stress under pro-atherogenic conditions.MethodsEndothelial cells were exposed to laminar shear stress (LSS) in a parallel-plate flow chamber containing oxidized low-density lipoprotein (oxLDL) in the perfusion solution, or remained static. We quantified the response of endothelial monolayers to LSS and oxLDL in terms of cell viability, barrier integrity, vascular endothelial cadherin (VE-cadherin) availability, focal adhesion (FA) remodeling, and monocyte-endothelial interactions.ResultsOur results showed that oxLDL stimulation and static conditions synergized to enhance endothelial barrier disruption. Under the same oxLDL challenge, the application of 25 dynes/cm2 LSS on the endothelial monolayer decreased the passage of fluorescein isothiocyanate (FITC)-dextran by 37.79%, increased transendothelial electrical resistance (TEER) by 24.97% compared with static cells (P<0.05), which was accompanied by reduced intercellular gap formation, relatively solid cell-substrate adhesion. Compared with static cells, endothelial cells exposed to both laminar flow and oxLDL had less small FAs, less monocyte adhesion and transmigration, and alleviated overexpression of VCAM-1 and MCP-1. Meanwhile, the oxLDL-induced internalization of VE-cadherin and vinculin were also attenuated by laminar flow, and this change was more pronounced at LSS of 25 dynes/cm2 than 5 dynes/cm2.ConclusionsStatic conditions favor, whereas physiologically higher levels of LSS ameliorate endothelial barrier disruption under pro-atherogenic stress, which is related to the improved availability of VE-cadherin and vinculin on the cell surface.

Myocardial infarction triggers Inflammatory deterioration of vascular niche by accelerating loss of a specific vessel subtype in bone

Abstract Introduction The interface between heart and bone emerges as a key trigger of post-infarction inflammation and progression of chronic heart failure (CHF). However, our knowledge on the underlying mechanisms of this interaction is incomplete. Bone vasculature, specifically so-called H-type (Endomucinhigh) endothelial cells (EC), plays a crucial role in maintenance of the bone integrity and regulation of hematopoietic stem cells (HSC). While previous studies in mice showed the reduction of H-type vessels by aging, the impact of ischemic heart disease is unclear. Therefore, we aimed to investigate the effects of myocardial infarction (MI) and chronic heart failure on the vascular bone cell composition in mice and humans. Methods and results Flow cytometric analysis of harvested bones at the different timepoints after MI induction in mice revealed a gradual loss of H-type endothelial cells in the time-course of developing chronic heart failure (P&amp;lt;0.05 at day 7 and 14 vs. control; P&amp;lt;0.0001 at day 28 vs. control). This results were confirmed in immunostainings of tibia sections showing a significant decrease of H-type vessel length after MI (p&amp;lt;0.05 at day 14 and 28, accordingly). The loss of type-H endothelium was accompanied by a significant expansion of long-term HSC in the bone (P=0.0005 at day 28 post MI vs. control). Importantly, type H ECs were also significantly reduced in the bone of ischemic post-infarct HF patients (n=16) compared with control subjects (n=8; P=0.0003). To gain insights into the mechanisms underlying the changes in the vascular niche, we performed single cells RNA sequencing of human BM ECs. These studies confirmed the decrease in Emcnexpressing ECs in ischemic HF patients, which was accompanied by significantly increased expression of inflammatory genes, including IL1b (P&amp;lt;0.0001). Inflammatory EC phenotypes and IL1b expression in HF could be further confirmed at protein level using cytospin immunostainings. Murine studies further revealed an early induction IL-1b specifically in H-type vessels already at day 1 after MI induction, which preceded the loss of H-type endothelium within the following 4 weeks. By inhibiting IL-1b production using a specific Nlrp3 inflammasome inhibitor (MCC950) we could observe a partial restoration of H-type EC frequencies (P=0.033) and a significant increase in H-type vessel length (p=0.035) at day 28 day after MI. Conclusions Our data show for the first time an impact of myocardial infarction and chronic heart failure on the bone marrow vasculature. These changes seem to be strongly associated with inflammatory response in H-type vessels, which precedes its loss after MI. Specifically, the induction of the inflammatory cytokine IL1b may contribute to the disturbed phenotype. This suggest that inhibition of IL1b (e.g. by canakinumab) be used as a novel strategy to prevent or reverse the deterioration of the vascular bone function in ischemic heart disease. Funding Acknowledgement Type of funding source: None

Impact of Wnt/β-catenin signaling on ethanol-induced changes in brain endothelial cell permeability.

Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). As previous studies suggested Wnt/β-catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17-200mM) for various periods of time (0-96hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50mM) caused a down-regulation of Wnt/β-catenin signaling, a reduction of tight junction protein expression and up-regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/β-catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3-6 fold increases in Dkk-1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2-fold compared to control). The ethanol-induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol-induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling.

Self-assembled VEGF-R2 targeting DNA aptamer-collagen fibers stimulate an angiogenic-like endothelial cell phenotype

Vascularization of engineered tissue is one of the hallmark challenges of tissue engineering. Leveraging self-assembled nucleic acid-collagen complexes (NACCs), we mixed a VEGF-R2 targeting aptamer or its receptor agonist divalent assembly with type I collagen to assemble NACC microfibers. Human umbilical vein endothelial cells (HUVECs) quickly remodeled these fibers into tubulogenic-like structures over 48 h. Moreover, NACCs made with the receptor agonist divalent aptamer assembly promoted enhanced expression of von Willebrand factor (vWF), angiopoietin-2 (ANGPT-2), and matrix metalloproteinase-2 (MMP-2) by HUVECs as measured by either immunocytochemistry or ELISA. The findings suggest, endothelial cell phenotype was directed by both biochemical cues afforded by the agonist behavior of the divalent aptamer assembly as well as by the biophysical cues afforded by the fibrous topography. Collectively, these results support the development of an angiogenic endothelial cell phenotype stimulated by the VEGF-R2 agonist NACC fibers. Thus, the combination of engineered DNA aptamer nanotechnology and DNA-collagen complexation phenomena is a promising biofunctional natural scaffold material system for tissue engineering and regenerative medicine applications.