EC Phenotype Research Articles

Abstract 14496: HIF2a Activation Reduces Pulmonary Angiogenesis and Promotes Vascular Remodeling in Pulmonary Arterial Hypertension by Suppressing Wnt7a Production in Pulmonary Endothelial Cells

Introduction: Dysfunctional angiogenesis and obliterative vasculopathy are hallmarks of pulmonary arterial hypertension (PAH). Inappropriate HIF1a/2a activation and insufficient Wnt7a production are associated with pulmonary artery endothelial cell (PAEC) dysfunction, suggesting that HIF1a/2a and Wnt7a may interact as part of a signaling axis to promote PAEC homeostasis. Hypothesis: HIF1a/2a overactivation suppresses Wnt7a expression in PAH PAECs. Methods: Healthy and PAH PMVECs were obtained from explanted lungs. Cobalt Chloride (CoCl2) was used to activate HIF. PMVECs were transfected with lentiviruses carrying HIF1a/2a constructs, followed by functional and molecular studies. Lungs from mice with endothelial-specific HIF1a/2a activation (Egln1 Tie2Cre ) and HIF2a deletion (EH2) were analyzed via single cell (sc) RNA-seq and confocal imaging. Results: Compared to controls, PAH lung tissue and PAECs demonstrated an inverse correlation between increased HIF1a/2a, and reduced Wnt7a expression. Treatment with CoCl2 for 24 hours resulted in a ~4-fold increase in Wnt7a transcription that was absent in PAH PMVECs. Lentivirus transfection of healthy PAEC demonstrated that HIF1a increased Wnt7a transcription while HIF2a significantly reduced Wnt7a transcription. In silico analysis of the Wnt7a gene locus demonstrated HIF binding sites at promoter-enhancer sequences suggesting that Wnt7a is a gene target of HIF signaling. In addition, ATAC-seq and ChIP-seq analysis of the Wnt7a gene locus in PAH PMVECs indicated a closed chromatin configuration, suggesting epigenetic repression. Lung scRNA-seq and RNAScope analysis of Egln1 Tie2Cre mice demonstrated a reduction of capillary EC, which was absent in EH2 mice, suggesting a HIF2a-dependent mechanism. Confocal and WB of lung tissues demonstrate that Wnt7a expression was significantly reduced in Egln1 Tie2Cre mice but preserved in EH2 mice. Conclusions: HIF2a and epigenetic repression contribute to PAEC dysfunction by suppressing Wnt7a-dependent angiogenesis in PAH. We speculate that Wnt7a may also be required to maintain the capillary EC phenotype in the lung and that restoring Wnt7a production could compensate for HIF-driven endothelial dysfunction in PAH.

Patient-specific and gene-corrected induced pluripotent stem cell-derived endothelial cells elucidate single-cell phenotype of pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension characterized by the preferential remodeling of the pulmonary venules. Hereditary PVOD is caused by biallelic variants of the EIF2AK4 gene. Three PVOD patients who carried the compound heterozygous variants of EIF2AK4 and two healthy controls were recruited and induced pluripotent stem cells (iPSCs) were generated from human peripheral blood mononuclear cells (PBMCs). The EIF2AK4 c.2965C>T variant (PVOD#1), c.3460A>T variant (PVOD#2), and c.4832_4833insAAAG variant (PVOD#3) were corrected by CRISPR-Cas9 in PVOD-iPSCs to generate isogenic controls and gene-corrected-iPSCs (GC-iPSCs). PVOD-iPSC-endothelial cells (ECs) exhibited a decrease in GCN2 protein and mRNA expression when compared with control and GC-ECs. PVOD-ECs exhibited an abnormal EC phenotype featured by excessive proliferation and angiogenesis. The abnormal phenotype of PVOD-ECs was normalized by protein kinase B inhibitors AZD5363 and MK2206. These findings help elucidate the underlying molecular mechanism of PVOD in humans and to identify promising therapeutic drugs for treating the disease.

X-box binding protein 1 as a key modulator in “healing endothelial cells”, a novel EC phenotype promoting angiogenesis after MCAO

BackgroundEndothelial cells (ECs) play an important role in angiogenesis and vascular reconstruction in the pathophysiology of ischemic stroke. Previous investigations have provided a profound cerebral vascular atlas under physiological conditions, but have failed to identify new disease-related cell subtypes. We aimed to identify new EC subtypes and determine the key modulator genes.MethodsTwo datasets GSE174574 and GSE137482 were included in the study. Seurat was utilized as the standard quality-control pipeline. UCell was used to calculate single-cell scores to validate cellular identity. Monocle3 and CytoTRACE were utilized in aid of pseudo-time differentiation analysis. CellChat was utilized to infer the intercellular communication pathways. The angiogenesis ability of ECs was validated by MTS, Transwell, tube formation, flow cytometry, and immunofluorescence assays in vitro and in vivo. A synchrotron radiation-based propagation contrast imaging was introduced to comprehensively portray cerebral vasculature.ResultsWe successfully identified a novel subtype of EC named “healing EC” that highly expressed pan-EC marker and pro-angiogenic genes but lowly expressed all the arteriovenous markers identified in the vascular single-cell atlas. Further analyses showed its high stemness to differentiate into other EC subtypes and potential to modulate inflammation and angiogenesis via excretion of signal molecules. We therefore identified X-box binding protein 1 (Xbp1) as a key modulator in the healing EC phenotype. In vitro and in vivo experiments confirmed its pro-angiogenic roles under both physiological and pathological conditions. Synchrotron radiation-based propagation contrast imaging further proved that Xbp1 could promote angiogenesis and recover normal vasculature conformation, especially in the corpus striatum and prefrontal cortex under middle cerebral artery occlusion (MCAO) condition.ConclusionsOur study identified a novel disease-related EC subtype that showed high stemness to differentiate into other EC subtypes. The predicted molecule Xbp1 was thus confirmed as a key modulator that can promote angiogenesis and recover normal vasculature conformation.

Abstract 15339: Er-Resident S1pr1 in Lung Endothelium Regulates Innate Immune Function and Mediates Inflammatory Lung Injury

Endothelial-cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is known to promote anti-inflammatory and barrier enhancing niche upon ligating S1P. Recently we showed that S1P and TNFα, later being a well-known inflammatory agonist, phosphorylate S1PR1 at tyrosine 143 (Y 143 ) which functions as an endoplasmic reticulum (ER) import signal (Anwar et al, 2021). ER-retained S1PR1 instructs barrier disruptive signaling but the mechanism remains unclear. Here, we generated S1PR1 knock-in mice using CRISPR-Cas9 strategy to edit endogenous S1PR1 into Y 143 D-S1PR1 (phospho mimicking) or Y 143 F-S1PR1 (phosphodefective) to test the hypothesis that ER-localized S1PR1 subverts EC from anti-inflammatory to pro-inflammatory EC leading to vascular injury. Because EC constitutes about 50% of cells in the lungs, we assessed if knock-in of Y 143 D-S1PR1 impaired lung homeostasis. We show that editing of S1PR1 into Y 143 D- or Y 143 F-S1PR1 did not alter total S1PR1 expression. Interestingly, Y 143 D-S1PR1 knock-in mice showed marked vascular leak at homeostasis along with increased neutrophil influx and inflammatory cytokine generation including TNFα, IL1ß and MiP2 as compared to Y 143 F-S1PR1 or WT mice. We next challenge these mice with intratracheal LPS. LPS-induced non-resolvable vascular inflammatory injury in Y 143 D-S1PR1 mice. Surprisingly, Y 143 F-S1PR1 knock-in mice did not develop vascular inflammatory injury. Furthermore, NFκB activity, a predominant transcription factor inducing inflammatory EC phenotype, was increased in EC transducing Y 143 D-S1PR1 mutant as compared to WT. However, TNFα failed to induce NFkB activity in EC transducing Y 143 F-S1PR1 mutant. Together, these results show that ER-resident S1PR1 program endothelial niche into immune-active niche by activating NFkB pathway leading to irreparable lung injury. Further experiments are being done to assess epigenetic changes (ATACseq and ChIP-Seq) in EC to address the concept the ER-resident S1PR1 controls the fate of immune cells in the lungs. We believe that understanding how ER-resident S1PR1 programs EC into inflammatory phenotype would allow development of new targets for treating the inflammatory vascular diseases including lung injury, ARDS, and COVID-19

Topography elicits distinct phenotypes and functions in human primary and stem cell derived endothelial cells

The effective deployment of arterial (AECs), venous (VECs) and stem cell-derived endothelial cells (PSC-ECs) in clinical applications requires understanding of their distinctive phenotypic and functional characteristics, including their responses to microenvironmental cues. Efforts to mimic the in-vivo vascular basement membrane milieu have led to the design and fabrication of nano- and micro-topographical substrates. Although the basement membrane architectures of arteries and veins are different, investigations into the effects of substrate topographies have so far focused on generic EC characteristics. Thus, topographical modulation of arterial- or venous-specific EC phenotype and function remains unknown. Here, we comprehensively evaluated the effects of 16 unique topographies on primary AECs, VECs and human PSC-ECs using a Multi Architectural (MARC) Chip. Gratings and micro-lenses augmented venous-specific phenotypes and depressed arterial functions in VECs; while AECs did not respond consistently to topography. PSC-ECs exhibited phenotypic and functional maturation towards an arterial subtype with increased angiogenic potential, NOTCH1 and Ac-LDL expression on gratings. Specific topographies could elicit different phenotypic and functional changes, despite similar morphological response in different ECs, demonstrating no direct correlation between the two responses.

Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis.

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.

HIV long-term non-progressors Elite Controllers: an Interplay Between host, Immune and Viral Factors

There is a rare group of HIV-1-infected individuals who show permanent control of clinical progression for over 10 years, maintain CD4+ cells >500 μl and have undetectable viral loads; they are designated long-term non-progressors elite controllers (LTNPs ECs). Multiple studies have demonstrated the necessary contribution of at least two of host, immune and viral factors to the LTNP phenotype. This group of individuals is not homogenous because of the different involvement of these factors. We will review the role of each of these and their combinations to the LTNP EC phenotype. LTNP EC individuals offer an opportunity for the investigation into the mechanisms for the spontaneous control of HIV infection.

31 - Phosphorylation of eNOS at Thr(T)495 induces mitochondrial fission and a hyper-proliferative endothelial cell phenotype in pulmonary arterial hypertension (PAH)

Mitochondrial dysfunction and cell proliferation play an important role in the pathological arterial constriction associated with pulmonary arterial hypertension (PAH). The mechanisms underlying this blood vessel remodeling is unclear. Recent studies have implicated a disruption of mitochondrial network dynamics to favor fission in the attenuation of mitochondrial function in PAH. We found that mitochondrial fission, or fragmentation, enhanced in endothelial cells isolated from IPAH patients. Further, this shift of morphology correlated with an increase of aerobic glycolysis, the Warburg effect. We also identified an increase in mitochondrial generated reactive oxygen species. These changes correlated with an increase in phospho(p)-T495eNOS protein levels Interestingly, high-resolution fluorescent imaging showed an increase in p-T495-eNOS levels on the mitochondria. We also found that the adenoviral-mediated overexpression of a phosphoT495eNOS mimic (T495DeNOS) in normal human pulmonary arterial endothelial cells (HPAEC) was sufficient to induce mitochondrial fission, a glycolytic shift, and stimulate proliferation. Mdivi-1, an inhibitor of the major mitochondrial fission regulator, Drp1 significantly attenuated both mitochondrial fission and cell proliferation. Finally, we found that inhibiting cellular glycolysis using the hexokinase 2 inhibitor, 2-deoxyglucose (2-DG), attenuated the proliferation of EC cultured form IPAH patients and sensitized them to TNF-α mediated apoptosis. Thus, we conclude that the phosphorylation of eNOS at T495 is intimately involved in regulating both mitochondrial morphology and the Warburg effect during PAH and the development of the hyper-proliferative anti-apoptotic EC phenotype associated with the development of PAH.

ZEB1 REGULATES MULTIPLE ASPECTS OF ENDOTHELIAL QUIESCENCE DURING VASCULAR REMODELLING

Generation of new blood vessels is essential for vascular remodeling in exercise to provide greater blood flow to developing muscles. As blood vessels grow, the sprouting endothelial tips need to recognise each other, and move from a motile, angiogenic phenotype to a maturing, quiescent phenotype,, form tubes and allow blood flow to occur and regulate their development. They grow in response to cytokine signalling such as VEGF and other angiogenic factors, but they mature as a consequence of cell contact and flow. As they do so, they change their metabolic state from growing, glycolytic, to quiescent oxidative metabolism. The mechanisms behind the transcriptional control of this phenotype are unknown, but recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) and expression of glycolytic enzymes such as PFKFB3 through c‐MYC. We investigated potential drivers of this process and identified Zeb1 (a transcription factor with known functions in cancer progression) to be expressed by quiescent (non‐angiogenic) endothelial cells and reduced in angiogenic endothelial cells in the developing murine retina. In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c‐myc and VEGFR2 and reduced FOXO1, Tie2 and VE‐cadherin expression. Consistently, Zeb1 knockdown EC were metabolically more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promoted EC migration and proliferation in vitro. In vivo shRNA knockdown of Zeb1 led to altered vessel formation and hypercellularity. In conclusion, Zeb1 appears to be linked to key quiescence regulating signaling pathways and furthermore regulates aspects of these pathways. Zeb1 expression appears to be a critical component for endothelial cells to generate functional vasculatures that can support enhanced muscle blood flow, by its control of FOXO1, PFKFB3 and Myc.Support or Funding InformationThis work was supported by the British Heart Foundation (grant PG/11/67/29067), the University of Nottingham Cancer Research Priority Area, and the Medical Research Council Newton Fund (MR/N01247X/1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 13521: Novel Protective Role of Cu Transporter ATP7A Against Cardiac Fibrosis by Inhibiting Endothelial-Mesenchymal Transition Mediated Through Snail and Antioxidant-1

Cardiac fibrosis is attributed to excess pathological deposition of extracellular-matrix (ECM) components; however, it is also mediated through endothelial-mesenchymal transition (EndMT) and fibroblast-myofibroblast transition (FMT) with unknown mechanisms. Copper (Cu), essential micronutrient, has been implicated in fibrosis. Bioavailability of Cu is tightly controlled by Cu-transporter ATP7A which obtains Cu from Cu chaperone Antioxidant-1 (Atox1), and delivers Cu to the ECM secretory Cu enzyme lysysl oxidase (LOX) or exclude Cu. Atox1 also functions as a Cu-dependent transcription factor. However, role of ATP7A in cardiac fibrosis remains unknown. Here we show that high dose angiotensin II infusion (740ng/kg/min for 10 days) to ATP7A mutant (ATP7Amut) mice with reduced Cu transport function significantly increased cardiac fibrosis (200%) and Cu (170%) compared to wild type mice. Blood pressure and heart/body weight were similar between each group. ATP7Amut mice showed increased Isolectin B4+/αSMA+ cells, indicating EndMT in fibrosis region, which was associated with increase in fibrosis-related genes (αSMA, TGFβ1/2/3, CTGF). In ATP7Amut fibroblasts, although LOX activity was reduced, phenotypic conversion to αSMA+ myofibroblasts (FMT) was accelerated. In ECs isolated from ATP7Amut mice or ATP7A-depleted HUVECs, EC phenotype was converted to mesenchymal phenotype with reduced expression of ECs markers (VE-cadherin, VEGFR2) and increased mesenchymal markers (αSMA, TGFβ2) and Cu levels. Functionally, conditioned media from ATP7A-depleted HUVECs promoted cardiac fibroblast differentiation to myofibroblasts (FMT). Mechanistically, ATP7A knockdown in ECs increased expression of EndMT master regulatory transcription factor Snail as well as Atox1 (2 fold). Atox1 overexpression by adenovirus increased EndMT-inducing genes (αSMA, TGFβ2, Hey2) via binding to their promoters at Atox1 binding sites (GAAAGA) without affecting EC markers or Snail which is shown to reduce EC markers. In summary, ATP7A limits cardiac fibrosis by inhibiting EndMT and FMT via suppressing transcription factors Snail and Atox1. Thus, ATP7A is a potential therapeutic target for fibrosis-dependent cardiovascular diseases.

Abstract 12454: Mitochondrial Transcription Factor 2B as a Mediator of Mitochondrial Retrograde Signaling and Endothelial Cell Shape

Mitochondrial dysfunction, such as observed in endothelial cells, has been implicated in various cardiovascular diseases including, hypertension and atherosclerosis. Mitochondrial transcription factor 2B (TFB2M) is an essential component to maintain proper transcriptional and functional control of mitochondrial DNA. As well, elongation of endothelial cells is a characteristic of atheroprotective regions within the vasculature, and the relationship between the mitochondria and EC shape is currently unknown. Additionally, recent interest has been focused on mechanisms by which the mitochondria may signal to the nucleus to affect cell function. The aim of our study is to investigate the hypothesis that TFB2M has a novel role in enhancing endothelial function. Human umbilical vein endothelial cells (HUVECs) were harvested 72 hours after adenoviral transduction with TFB2M (100 moi). HUVECs transduced with TFB2M showed an elongated cell morphology when compared to GFP control. To further investigate the effect of TFB2M on regulating mitochondrial function and cell shape, immunoblotting was carried out for markers involved in mitochondrial function/dynamics and markers indicative of cytoskeleton reorganization. TFB2M transduction resulted in increased expression of mitochondrial biogenesis marker VDAC (2.6 fold increase), mitochondrial fusion protein MFN2 (2.1 fold increase), and phosphorylated myosin phosphatase targeting protein MYPT1 at Thr850 (2.2 fold increase, p < 0.05 for all proteins). Additionally, fluorescence microscopy showed enhanced mitochondrial fluorescence in TFB2M transduced cells using mitotracker red staining (3.5 fold increase, p < 0.001). These data indicate that TFB2M has a previously undiscovered function contributing to altered EC function and shape, potentially through a novel mitochondrial retrograde signaling mechanism. Further research will focus on distinguishing the exact mechanisms culminating in a protective EC phenotype and the beneficial role of endothelial TFB2M-mediated enhanced mitochondrial function in the treatment of EC dysfunction associated with various cardiovascular diseases.

Abstract 17231: Contact-Mediated Interaction Between Pulmonary Artery Endothelial and Smooth Muscle Cells Promotes a BMPR2-β-catenin-Notch1 Signal Causing Hyperpolarization of Endothelial Mitochondria and a Stalk Cell-like Phenotype

Background: Pulmonary arterial hypertension (PAH) is characterized by endothelial (EC) apoptosis related to loss of distal (D) pulmonary arteries (PA) and abnormal proliferation of smooth muscle-like cells (SMC). These features are mediated by impaired cell metabolism. We observed mitochondrial dysfunction in human PAEC with reduced or mutant BMPR2, the gene associated with familial PAH. Deletion of BMPR2 in murine EC causes failure to regenerate DPA and to recover from hypoxia induced pulmonary hypertension. Loss of BMPR2 in PASMC results in mitochondrial dysfunction associated with enhanced proliferation. Since metabolites and other factors are often transferred by cell contact, we investigated how PASMC-PAEC contact impacts PAEC mitochondrial and cellular function, and could be perturbed by a BMPR2 mutation in PAEC and/or PASMC. Methods and Results: We used a co-culture system in which PAEC and PASMC from lungs of donor control (c-) or PAH patients with a BMPR2 mutation (m-) were either seeded on opposite sides of a 1μm pore insert or separated to assess non-contact mediated responses. When c-PAEC contacted c-PASMC, PAEC mitochondrial membrane potential (Δψ) increased 6-fold compared to mono-cultured or non-contact co-cultured cells (p < 0.001). These PAEC exhibited a significant increase in proliferation (3-fold increase by Ki-67 staining, p<0.01) and survival (73% reduction in caspase activity, p < 0.001); however we observed a decrease in migration and angiogenic capacity in modified Boyden chamber and matrigel assays (52% and 60% reduction, p < 0.001, respectively). We related the increase in Δψ and the above ‘stalk cell-like’ phenotype to an increase in Notch1 intracellular domain (2.5-fold increase, p < 0.001), as they were abrogated by the gamma secretase inhibitor, and enhanced by the co-transcriptional activator β-catenin (4-fold increase, p < 0.001). When m-PAEC and m-PASMC were used in contact cultures, these features were not observed. Conclusions: PASMC in contact with PAEC increase Δψ and produce a Notch and β-catenin mediated stalk cell-like EC phenotype. These features are lost when BMPR2 is deficient in both PAEC and PASMC suggesting that BMPR2-mediated PASMC-EC contact allows PAEC to survive and regenerate in response to damage.

Abstract P122: Mitochondrial Transcription Factor 2B Regulates Endothelial Cell Morphology

Mitochondrial dysfunction, such as observed in endothelial cells, has been implicated in various cardiovascular diseases including, hypertension and atherosclerosis. Mitochondrial transcription factor 2B (TFB2M) is an essential component to maintain proper transcriptional and functional control of mitochondrial DNA. As well, elongation of endothelial cells is a characteristic of atheroprotective regions within the vasculature, and the relationship between the mitochondria and EC shape is currently unknown. The aim of our study is to investigate the hypothesis that TFB2M has a novel role in enhancing endothelial function. Human umbilical vein endothelial cells (HUVECs) were harvested 72 hours after adenoviral transduction with TFB2M (100 moi). HUVECs transduced with TFB2M showed an elongated cell morphology when compared to GFP control. To further investigate the effect of TFB2M on regulating mitochondrial function and cell shape, immunoblotting was carried out for markers involved in mitochondrial function/dynamics and markers indicative of cytoskeleton reorganization. TFB2M transduction resulted in increased expression of mitochondrial biogenesis marker VDAC (2.6 fold increase), mitochondrial fusion protein MFN2 (2.1 fold increase), and phosphorylated myosin phosphatase targeting protein MYPT1 at Thr850 (2.2 fold increase, p < 0.05 for all proteins). Additionally, fluorescence microscopy showed enhanced mitochondrial fluorescence in TFB2M transduced cells using mitotracker red staining (3.5 fold increase, p < 0.001). These data indicate that TFB2M has a previously undiscovered function contributing to altered EC function and shape, potentially through a novel mitochondrial retrograde signaling mechanism. Further research will focus on distinguishing the exact mechanisms culminating in a protective EC phenotype and the beneficial role of endothelial TFB2M-mediated enhanced mitochondrial function in the treatment of EC dysfunction associated with various cardiovascular diseases.

Abstract 12495: Fibrin-Thrombin Patch for Endothelial Cell Differentiation and Cardiac-Tissue Patch Manufacturing Using Human Induced-Pluripotent Stem Cells

Aim: To explore the feasibility using fibrin-thrombin patch for endothelial cell differentiation and cardiac scaffold manufacturing using cardiac cells derived from human induced-pluripotent stem cells (hiPSCs). Method and Result: hiPSCs were dissociated into single cells and seeded into three-dimensional (3D) fibrin-thrombin patches and undergo a two-stage differentiation protocol. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. A human cardiac-tissue patch (hiCP) was developed by seeding the hiPSC-ECs with hiPSC-derived cardiomyocytes (hiPSC-CMs) and smooth muscle cells (hiPSC-SMCs) into a 3D fibrin scaffold. The hiCP began to contract 3 days after synthesis, 4 days earlier than patches that were created identically but without hiPSC-ECs, and continued to beat regularly (100-120 beats/min) for at least 4 weeks in vitro. Conclusion: These data demonstrate that this new 3D differentiation protocol can efficiently generate stable ECs from hiPSCs and, furthermore, that the differentiated hiPSC-ECs can be combined with hiPSC-CMs and -SMCs to construct an hiCP with improved contractile activity. Our observations also suggest that interactions between the cardiac endothelium and myocytes may contribute to the optimized beating of hiCPs.

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Phenotypic characteristics of pulmonary supernumerary arterial endothelium may be involved in the formation of plexiform lesions (1089.21)

The plexiform lesion (PL), which comprises proliferative endothelial marker (EC)‐positive channel cells and chromatin‐rich core cells, is the histological hallmark of severe pulmonary arterial hypertension (PAH). PLs preferentially form in supernumerary arteries (SAs) in SU5416/hypoxia/normoxia‐exposed PAH rats, which supports clinical observations. However, the underlying pathological mechanisms of this observation are unknown. SAs are unique small pulmonary branches with specific anatomical and functional properties, such as the phenotype of the ECs, which may contribute to the formation of PLs. The EC phenotype in PLs seems to be shared with pulmonary microvascular ECs (PMVECs), which are highly proliferative. Therefore, we tested the hypothesis that the ECs in SAs share the same phenotype as PMVECs that predisposes to the development of PLs.A lectin protein from Griffonia simplicifolia (GS), which selectively interacts with PMVECs was perfused through the pulmonary vasculature of control and PAH rats. Lung slices were prepared and examined by confocal fluorescence microscopy.In normal rats, ECs in SAs interacted with GS. However, ECs in SAs and occlusive vascular lesions in PAH rats had no apparent interaction with GS.The PMVEC‐like phenotype of ECs in SAs may contribute to the development of PLs, and there may be a phenotypic transformation of these cells during the progression of PAH.Grant Funding Source: Supported by NIH HL66299 and HL60024 to T.S.

The influence of a spatiotemporal 3D environment on endothelial cell differentiation of human induced pluripotent stem cells

Current EC differentiation protocols are inefficient, and the phenotypes of the differentiated ECs are only briefly stable, which significantly inhibits their utility for basic science research. Here, a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively, these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and, furthermore, the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks in vitro.

Resveratrol promotes endothelial cell wound healing under laminar shear stress through an estrogen receptor-α-dependent pathway

Restenosis is an adverse outcome of angioplasty, characterized by vascular smooth muscle cell (VSMC) hyperplasia. However, therapies targeting VSMC proliferation delay re-endothelialization, increasing the risk of thrombosis. Resveratrol (RESV) inhibits restenosis and promotes re-endothelialization after arterial injury, but in vitro studies assessing RESV-mediated effects on endothelial cell growth contradict these findings. We thus hypothesized that fluid shear stress, mimicking physiological blood flow, would recapitulate RESV-dependent endothelial cell wound healing. Since RESV is an estrogen receptor (ER) agonist, we tested whether RESV promotes re-endothelialization through an ER-α-dependent mechanism. Mice fed a high-fat diet or a diet supplemented with RESV were subjected to carotid artery injury. At 7 days after injury, RESV significantly accelerated re-endothelialization compared with vehicle. In vitro wound healing assays demonstrated that RESV exhibits cell-type selectivity, inhibiting VSMC, but not endothelial cell growth. Under laminar shear stress (LSS), RESV dramatically enhanced endothelial cell wound healing and increased both the activation of extracellular signal-regulated kinase (ERK) and endothelial cell proliferation. Under LSS, small interfering RNA against ER-α, but not endothelial nitric oxide synthase, abolished RESV-induced ERK activation, endothelial cell proliferation, and wound healing. Thus these studies suggest that the EC phenotype induced by LSS better models the prohealing effects of RESV and that RESV and LSS interact to promote an ER-α-dependent mitogenic effect in endothelial cells.

Abstract 222: Dkk1 and Msx2-Wnt7b Signaling Reciprocally Regulate the Endothelial-Mesenchymal Transition in Aortic Endothelial Cells

Objective Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition (EndMT) during tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic calcification and fibrosis. We studied the impact of Wnt7, Msx2, and Dkk1 (Wnt7 antagonist) on EndMT in primary aortic endothelial cells (AoECs). Methods and Results Transduction of AoECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1 and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibrogenic and osteogenic markers - e.g., SM22, type I collagen, Osx, Runx2, alkaline phosphatase – were upregulated by Dkk1 via activin-like kinase / Smad pathways. Dkk1 increased fibrosis and mineralization of AoECs cultured under osteogenic conditions - the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained the “cobblestone” morphology of differentiated ECs and promoted EC marker expression. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR-/- mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, Msx2) and nuclear pSmad1/5, and increased collagen accumulation. Conclusions Dkk1 enhances EndMT in AoECs, while Msx2-Wnt7 signals stabilize EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal cell responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.

High-level Shear Stress Stimulates Endothelial Differentiation and VEGF Secretion by Human Mesenchymal Stem Cells

Growing experimental evidence suggests that mechanical stimulation play important roles in determining the proliferation, migration, and apoptosis of human mesenchymal stem cells (hMSCs). Here, we show that shear stress stimulates hMSCs toward an EC phenotype in the absence of chemical induction. Most importantly, fluorescence microscopy clearly demonstrated for the first time that the distributions of endothelial-specific markers, vascular endothelial (VE)-cadherin and CD31, in hMSCs were similar to those of ECs at cell–cell adhesion sites after exposing hMSCs to a shear stress of 2 Pa for 2 days with subsequent static culture for 5 days. Western blot analysis proved that shear stress of 2 Pa significantly induced protein expression of von Willebrand factor (vWF), VE–cadherin, and CD31. However, an unclear expression of the endothelial-specific markers was observed in the 0.2 Pa shear stress group. In addition, there was a cumulative production of vascular endothelial growth factor (VEGF), which is known to induce endothelial differentiation of MSCs. By exerting shear stress of 2 Pa on hMSCs for 2 days with subsequent culture for 5 days, the production level of approximately 2-fold compared with that of the control group was achieved. Our findings suggest that high-level shear stress can induce VEGF production and EC differentiation from hMSCs. This may provide a means for addressing the cell sourcing issue for effective tissue engineering.