Incremental Experience in In Vitro Primary Culture of Human Pulmonary Arterial Endothelial Cells Harvested from Swan-Ganz Pulmonary Arterial Catheters

Pulmonary arterial hypertension (PAH) is a devastating condition affecting the pulmonary microvascular wall and endothelium, resulting in their partial or total obstruction. Despite a combination of expensive vasodilatory therapies, mortality remains high. Personalized therapeutic approaches, based on access to patient material to unravel patient specificities, could move the field forward. An innovative technique involving harvesting pulmonary arterial endothelial cells (PAECs) at the time of diagnosis was recently described. The aim of the present study was to fine-tune the initial technique and to phenotype the evolution of PAECs in vitro subcultures. PAECs were harvested from Swan-Ganz pulmonary arterial catheters during routine diagnostic or follow up right heart catheterization. Collected PAECs were phenotyped by flow cytometry and immunofluorescence focusing on endothelial-specific markers. We highlight the ability to harvest patients’ PAECs and to maintain them for up to 7–12 subcultures. By tracking the endothelial phenotype, we observed that PAECs could maintain an endothelial phenotype for several weeks in culture. The present study highlights the unique opportunity to obtain homogeneous subcultures of primary PAECs from patients at diagnosis and follow-up. In addition, it opens promising perspectives regarding tailored precision medicine for patients suffering from rare pulmonary vascular diseases.

Induced endothelial cells from peripheral arterial disease patients and neonatal fibroblasts have comparable angiogenic properties

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.

Abstract P738: Matrix Metalloproteinase-3 (mmp3) Activation Impairs Endothelial Integrity and Increases Vascular Injury After Ischemic Stroke in Female Diabetic Rats

Introduction: Females patients with diabetes suffer from poor outcomes of ischemic stroke but underlying reasons are not fully understood. We have shown that 1) adult female diabetic rats develop greater infarct and hemorrhagic transformation (HT) leading to poorer outcomes after ischemic stroke, and 2) MMP3 activity is increased to a greater extent in female endothelial cells as well as the microvasculature of diabetic female rats than in males. This led us to hypothesize that MMP3 mediates disruption of endothelial integrity amplifying vascular injury in female diabetic rats. Methods: Diabetic female Wistar rats, subjected to 60 min MCAO, received a single dose of MMP3 inhibitor (UK356618; 15mg/kg; iv) or vehicle after reperfusion. On Day 3, adhesive removal time (ART), behavioral composite score, brain infarct size, edema and macroscopic HT were recorded. Primary brain microvascular endothelial cells (BMVECs) isolated from female rats were cultured in 25mM glucose plus 100μM sodium palmitate for 48 hours followed by hypoxia insult for 24 hours in the presence/absence of UK356618 (20nM). Endothelial phenotype and integrity were assessed. Results: Brain edema and HT scores were lower and outcomes were improved with MMP3 inhibition but infarct size was not different between the groups (Table). Hypoxia decreased tight junction protein occludin-1 while increasing transforming growth factor receptor-1 (TGF-R1), a key modulator of endothelial phenotype. Treatment with inhibitor UK356618 reversed these responses. Conclusions: The lack of a difference in infarct size with the treatment suggests that MMP3 inhibition improves short term outcomes in female diabetic rats via preservation of endothelial integrity. The decrease in TGF-R1 needs to be further investigated for long term effects of MMP3 inhibition on vascular restoration and recovery after stroke in diabetes.

Abstract 16966: A p53-Toll-Like Receptor 3-Bone Morphogenic Protein Interaction Regulates the Endothelial Phenotype and Function in Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by complex remodeling of pulmonary arteries. In addition to impaired bone morphogenic protein (BMP) signaling, deficiency of the cell cycle regulator p53 has been implicated in PAH pathogenesis. Endothelial cells (ECs) from PAH patients have reduced levels of toll-like receptor 3 (TLR3), impairing EC function. This study investigated whether the p53, TLR3 and BMP pathways regulate endothelial phenotype and function in PAH. Methods: Human pulmonary artery ECs (PAECs) and rat CD117 + ECs were used. Knockdown experiments were carried out by targeting p53 and TLR3. Cells were further treated with the MDM2 antagonist Nutlin 3a, reducing p53 degradation, and with the TLR3 agonist Poly(I:C). Rats received CD117 + EC clones or SU5416 and were subjected to chronic hypoxia and then treated with Nutlin-3a, Poly(I:C) or vehicle. Results: We detected reduced p53 levels in PAECs from PAH patients. Clonal expansion of rat CD117 + ECs resulted in hyperproliferative cells with reduced p53 and TLR3 levels and evidence for histone methylation. Then, knockdown of p53 or TLR3 increased tube formation in surviving human and rat ECs. In addition, knockdown or activation of p53 indicated regulation of TLR3 mRNA by p53. TLR3 knockdown reduced expression of BMP2, whereas the TLR3 agonist Poly(I:C) induced BMP receptor 2 expression. Upstream, reduced p53 degradation by Nutlin 3a not only increased TLR3, but also BMP2 expression and increased BMP signaling in rat ECs. Interestingly, clonal selection resulted in EC clones with high BMP2 expression. TLR3 knockdown caused partial endothelial mesenchymal transition with increased expression of Snail, which was ameliorated by Poly(I:C). In vivo, Nutlin-3a treatment reduced PH while increasing TLR3 expression in rats. Poly(I:C) reduced PH and Smad2 phosphorylation in rats, and the protective effects were abolished in TRIF KO mice. Conclusion: The RNA receptor and p53 transcriptional target TLR3 regulates the BMP pathway in ECs and this interaction may explain some of the protective effects of TLR3 stimulation in PH. However as clonally selected ECs retain high BMP2 expression, differences between EC subpopulations can be suggested and are under investigation.

2980The effect of monoclonal antibodies against PCSK9 on circulating CD34+ progenitor cells interactions with platelets in patients with familial hypercholesterolemia

Introduction Platelets interact with circulating CD34+ progenitor cells inducing their differentiation into endothelial progenitor cells (EPCs) and further promoting EPC maturation into endothelial cells, an important step for endothelial regeneration and angiogenesis. PSCK9 is a serine protease that may not only play a crucial role in binding to the low density lipoprotein receptor (LDL-R) increasing its endosomal and lysosomal degradation thus inhibiting its recycling to the cell surface, leading to increasing LDL-cholesterol levels in plasma, but may also exhibit several, independent on LDL-R, activities on a plethora of cell types, including platelets. Purpose We investigated whether monoclonal antibodies (mabs) against PCSK9 could affect platelet interaction with CD34+ cells as well as their differentiation into EPCs, in patients with Familial Hypercholestorelemia (FH). Patients and methods Patients with FH (n=11, 8 men and 3 women, mean age 53±10 years) being under statin/ezetimibe therapy (40mg statin/ 10mg ezetimibe) participated in the study. Patients exhibited resisting high LDL-cholesterol levels, therefore following the existing guidelines, they received mab against PCSK9 (7 evolocumab, 140mg/ml and 4 alirocumab, 150mg/ml) every two weeks. Blood samples were drawn before and after 4 doses of the antibody i.e. after two months (Follow-up). In addition to the patients' serum lipid profile, the endothelial phenotype of CD34+ (membrane expression levels of KDR which is a receptor for VEGF and an endothelial phenotype marker) on CD34+ cells (CD34+-KDR+ cells) and the formation of platelet-CD34+ (CD61+-CD34+) and platelet-KDR+ (CD61+-KDR+) conjugates were also determined by flow cytometry on whole blood after dual labelling with anti-VEGFR-R2/KDR-PE, anti-CD61-PerCP and anti-CD34-FITC. The results were expressed as the %gated of CD34+-KDR+ cells, as well as CD61+-CD34+ and CD61+-KDR+ conjugates. Results The baseline LDL-cholesterol levels were significantly reduced from 172±43 mg/dl to 51±17 at follow-up, p=0.0001. Importantly, the formation of CD61+-CD34+ and CD61+-KDR+ conjugates were increased from baseline to follow-up (from 1.39±1.3 to 2.23±2.8, p=0.037) and (from 2.91±4.2 to 5.92±6.01, p=0.014), respectively. The membrane expression of KDR on CD34+ cells was also increased from 0.79±0.9 to 1.36±0.90, p=0.042, which reflects the increase in the CD34+ endothelial phenotype. Conclusion We show for the first time that the mabs against PCSK9, significantly increase the CD34+ endothelial phenotype in FH patients, which may at least partially attributed to the increase in the platelet interaction with CD34+ cells (platelet-CD34+ conjugates). The underlying mechanisms as well as the consequences of this effect at the clinical level for these patients are under investigation.