Epigenetic Modification of the von Willebrand Factor Promoter Drives Platelet Aggregation on the Pulmonary Endothelium in Chronic Thromboembolic Pulmonary Hypertension.

Rationale: von Willebrand factor (vWF) mediates platelet adhesion during thrombosis. While chronic thromboembolic pulmonary hypertension (CTEPH) is associated with increased plasma levels of vWF, the role of this protein in CTEPH has remained enigmatic. Objectives: To identify the role of vWF in CTEPH. Methods: CTEPH-specific patient plasma and pulmonary endarterectomy material from patients with CTEPH were used to study the relationship between inflammation, vWF expression, and pulmonary thrombosis. Cell culture findings were validated in human tissue, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Measurements and Main Results: vWF is increased in plasma and the pulmonary endothelium of CTEPH patients. In vitro, the increase in vWF gene expression and the higher release of vWF protein upon endothelial activation resulted in elevated platelet adhesion to CTEPH endothelium. Proteomic analysis revealed that nuclear factor (NF)-κB2 was significantly increased in CTEPH. We demonstrate reduced histone tri-methylation and increased histone acetylation of the vWF promoter in CTEPH endothelium, facilitating binding of NF-κB2 to the vWF promoter and driving vWF transcription. Genetic interference of NFκB2 normalized the high vWF RNA expression levels and reversed the prothrombotic phenotype observed in CTEPH-pulmonary artery endothelial cells. Conclusions: Epigenetic regulation of the vWF promoter contributes to the creation of a local environment that favors in situ thrombosis in the pulmonary arteries. It reveals a direct molecular link between inflammatory pathways and platelet adhesion in the pulmonary vascular wall, emphasizing a possible role of in situ thrombosis in the development or progression of CTEPH.

Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Exacerbated inflammatory signaling underlies aberrant response to BMP9 in pulmonary arterial hypertension lung endothelial cells.

Imbalanced transforming growth factor beta (TGFß) and bone morphogenetic protein (BMP) signaling are postulated to favor a pathological pulmonary endothelial cell (EC) phenotype in pulmonary arterial hypertension (PAH). BMP9 is shown to reinstate BMP receptor type-II (BMPR2) levels and thereby mitigate hemodynamic and vascular abnormalities in several animal models of pulmonary hypertension (PH). Yet, responses of the pulmonary endothelium of PAH patients to BMP9 are unknown. Therefore, we treated primary PAH patient-derived and healthy pulmonary ECs with BMP9 and observed that stimulation induces transient transcriptional signaling associated with the process of endothelial-to-mesenchymal transition (EndMT). However, solely PAH pulmonary ECs showed signs of a mesenchymal trans-differentiation characterized by a loss of VE-cadherin, induction of transgelin (SM22α), and reorganization of the cytoskeleton. In the PAH cells, a prolonged EndMT signaling was found accompanied by sustained elevation of pro-inflammatory, pro-hypoxic, and pro-apoptotic signaling. Herein we identified interleukin-6 (IL6)-dependent signaling to be the central mediator required for the BMP9-induced phenotypic change in PAH pulmonary ECs. Furthermore, we were able to target the BMP9-induced EndMT process by an IL6 capturing antibody that normalized autocrine IL6 levels, prevented mesenchymal transformation, and maintained a functional EC phenotype in PAH pulmonary ECs. In conclusion, our results show that the BMP9-induced aberrant EndMT in PAH pulmonary ECs is dependent on exacerbated pro-inflammatory signaling mediated through IL6.

Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Multicenter Preclinical Validation of BET Inhibition for the Treatment of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is a degenerative arteriopathy that leads to right ventricular (RV) failure. BRD4 (bromodomain-containing protein 4), a member of the BET (bromodomain and extra-terminal motif) family, has been identified as a critical epigenetic driver for cardiovascular diseases.Objectives: To explore the therapeutic potential in PAH of RVX208, a clinically available BET inhibitor.Methods: Microvascular endothelial cells, smooth muscle cells isolated from distal pulmonary arteries of patients with PAH, rats with Sugen5416 + hypoxia- or monocrotaline + shunt-induced PAH, and rats with RV pressure overload induced by pulmonary artery banding were treated with RVX208 in three independent laboratories.Measurements and Main Results: BRD4 is upregulated in the remodeled pulmonary vasculature of patients with PAH, where it regulates FoxM1 and PLK1, proteins implicated in the DNA damage response. RVX208 normalized the hyperproliferative, apoptosis-resistant, and inflammatory phenotype of microvascular endothelial cells and smooth muscle cells isolated from patients with PAH. Oral treatment with RVX208 reversed vascular remodeling and improved pulmonary hemodynamics in two independent trials in Sugen5416 + hypoxia-PAH and in monocrotaline + shunt-PAH. RVX208 could be combined safely with contemporary PAH standard of care. RVX208 treatment also supported the pressure-loaded RV in pulmonary artery banding rats.Conclusions: RVX208, a clinically available BET inhibitor, modulates proproliferative, prosurvival, and proinflammatory pathways, potentially through interactions with FoxM1 and PLK1. This reversed the PAH phenotype in isolated PAH microvascular endothelial cells and smooth muscle cells in vitro, and in diverse PAH rat models. RVX208 also supported the pressure-loaded RV in vivo. Together, these data support the establishment of a clinical trial with RVX208 in patients with PAH.

MnTBAP Reverses Pulmonary Vascular Remodeling and Improves Cardiac Function in Experimentally Induced Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by obstructed pulmonary vasculatures. Current therapies for PAH are limited and only alleviate symptoms. Reduced levels of BMPR2 are associated with PAH pathophysiology. Moreover, reactive oxygen species, inflammation and autophagy have been shown to be hallmarks in PAH. We previously demonstrated that MnTBAP, a synthetic metalloporphyrin with antioxidant and anti-inflammatory activity, inhibits the turn-over of BMPR2 in human umbilical vein endothelial cells. Therefore, we hypothesized that MnTBAP might be used to treat PAH. Human pulmonary artery endothelial cells (PAECs), as well as pulmonary microvascular endothelial (MVECs) and smooth muscle cells (MVSMCs) from PAH patients, were treated with MnTBAP. In vivo, either saline or MnTBAP was given to PAH rats induced by Sugen 5416 and hypoxia (SuHx). On PAECs, MnTBAP was found to increase BMPR2 protein levels by blocking autophagy. Moreover, MnTBAP increased BMPR2 levels in pulmonary MVECs and MVSMCs isolated from PAH patients. In SuHx rats, MnTBAP reduced right ventricular (RV) afterload by reversing pulmonary vascular remodeling, including both intima and media layers. Furthermore, MnTBAP improved RV function and reversed RV dilation in SuHx rats. Taken together, these data highlight the importance of MnTBAP as a potential therapeutic treatment for PAH.

Contribution of Impaired Parasympathetic Activity to Right Ventricular Dysfunction and Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). METHODS: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (α-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. RESULTS: Patients with PAH with lower RV ejection fraction (<41%) had a significantly reduced heart rate recovery in comparison with patients with higher RV ejection fraction. In PAH RV samples, α-7 nicotinic acetylcholine receptor was increased and acetylcholinesterase activity was reduced versus controls. No difference in muscarinic acetylcholine type 2 receptor expression was observed. Chronic PYR treatment in PH rats normalized the cardiovascular autonomic function, demonstrated by an increase in parasympathetic activity and baroreflex sensitivity. PYR improved survival, increased RV contractility, and reduced RV stiffness, RV hypertrophy, RV fibrosis, RV inflammation, and RV α-7 nicotinic acetylcholine receptor and muscarinic acetylcholine type 2 receptor expression, as well. Furthermore, PYR reduced pulmonary vascular resistance, RV afterload, and pulmonary vascular remodeling, which was associated with reduced local and systemic inflammation. CONCLUSIONS: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.

Prevention of progression of pulmonary hypertension by the Nur77 agonist 6-mercaptopurine: role of BMP signalling.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease characterised by abnormal remodelling of pulmonary vessels, leading to increased vascular resistance and right ventricle failure. This abnormal vascular remodelling is associated with endothelial cell dysfunction, increased proliferation of smooth muscle cells, inflammation and impaired bone morphogenetic protein (BMP) signalling. Orphan nuclear receptor Nur77 is a key regulator of proliferation and inflammation in vascular cells, but its role in impaired BMP signalling and vascular remodelling in PAH is unknown.We hypothesised that activation of Nur77 by 6-mercaptopurine (6-MP) would improve PAH by inhibiting endothelial cell dysfunction and vascular remodelling.Nur77 expression is decreased in cultured pulmonary microvascular endothelial cells (MVECs) and lungs of PAH patients. Nur77 significantly increased BMP signalling and strongly decreased proliferation and inflammation in MVECs. In addition, conditioned medium from PAH MVECs overexpressing Nur77 inhibited the growth of healthy smooth muscle cells. Pharmacological activation of Nur77 by 6-MP markedly restored MVEC function by normalising proliferation, inflammation and BMP signalling. Finally, 6-MP prevented and reversed abnormal vascular remodelling and right ventricle hypertrophy in the Sugen/hypoxia rat model of severe angioproliferative PAH.Our data demonstrate that Nur77 is a critical modulator in PAH by inhibiting vascular remodelling and increasing BMP signalling, and activation of Nur77 could be a promising option for the treatment of PAH.

Microcirculatory perfusion disturbances following cardiac surgery with cardiopulmonary bypass are associated with in vitro endothelial hyperpermeability and increased angiopoietin-2 levels.

BACKGROUND: Endothelial hyperpermeability following cardiopulmonary bypass (CPB) contributes to microcirculatory perfusion disturbances and postoperative complications after cardiac surgery. We investigated the postoperative course of renal and pulmonary endothelial barrier function and the association with microcirculatory perfusion and angiopoietin-2 levels in patients after CPB. METHODS: Clinical data, sublingual microcirculatory data, and plasma samples were collected from patients undergoing coronary artery bypass graft surgery with CPB (n = 17) before and at several time points up to 72 h after CPB. Renal and pulmonary microvascular endothelial cells were incubated with patient plasma, and in vitro endothelial barrier function was assessed using electric cell-substrate impedance sensing. Plasma levels of angiopoietin-1,-2, and soluble Tie2 were measured, and the association with in vitro endothelial barrier function and in vivo microcirculatory perfusion was determined. RESULTS: A plasma-induced reduction of renal and pulmonary endothelial barrier function was observed in all samples taken within the first three postoperative days (P < 0.001 for all time points vs. pre-CPB). Angiopoietin-2 and soluble Tie2 levels increased within 72 h after CPB (5.7 ± 4.4 vs. 1.7 ± 0.4 ng/ml, P < 0.0001; 16.3 ± 4.7 vs. 11.9 ± 1.9 ng/ml, P = 0.018, vs. pre-CPB), whereas angiopoietin-1 remained stable. Interestingly, reduced in vitro renal and pulmonary endothelial barrier moderately correlated with reduced in vivo microcirculatory perfusion after CPB (r = 0.47, P = 0.005; r = 0.79, P < 0.001). In addition, increased angiopoietin-2 levels moderately correlated with reduced in vitro renal and pulmonary endothelial barrier (r = - 0.46, P < 0.001; r = - 0.40, P = 0.005) and reduced in vivo microcirculatory perfusion (r = - 0.43, P = 0.01; r = - 0.41, P = 0.03). CONCLUSIONS: CPB is associated with an impairment of in vitro endothelial barrier function that continues in the first postoperative days and correlates with reduced postoperative microcirculatory perfusion and increased circulating angiopoietin-2 levels. These results suggest that angiopoietin-2 is a biomarker for postoperative endothelial hyperpermeability, which may contribute to delayed recovery of microcirculatory perfusion after CPB. TRIAL REGISTRATION: NTR4212 .

The covalently immobilized antimicrobial peptide LL37 acts as a VEGF mimic and stimulates endothelial cell proliferation.

The chemical coupling of growth factors to solid substrates are discussed as an alternative to delivery systems. Utilizing entire proteins for this application is hampered by safety and stability considerations. Instead, growth factor mimicking peptides are of great interest for biomedical applications, such as tissue engineering, due to their purity and stability. The human cathelicidin derived antimicrobial peptide LL37, beside its microbicidal activity, was shown to stimulate endothelial cell growth when used in a soluble form. Here, in a novel approach, spacer mediated immobilization, but not direct conjugation of LL37, to a gold substrate was shown to result in a pronounced mitogenic effect on endothelial cells, comparable to that of soluble vascular endothelial growth factor.

Blood Outgrowth and Proliferation of Endothelial Colony Forming Cells are Related to Markers of Disease Severity in Patients with Pulmonary Arterial Hypertension.

In pulmonary arterial hypertension (PAH), lung-angioproliferation leads to increased pulmonary vascular resistance, while simultaneous myocardial microvessel loss contributes to right ventricular (RV) failure. Endothelial colony forming cells (ECFC) are highly proliferative, angiogenic cells that may contribute to either pulmonary vascular obstruction or to RV microvascular adaptation. We hypothesize ECFC phenotypes (outgrowth, proliferation, tube formation) are related to markers of disease severity in a prospective cohort-study of 33 PAH and 30 healthy subjects. ECFC were transplanted in pulmonary trunk banded rats with RV failure. The presence of ECFC outgrowth in PAH patients was associated with low RV ejection fraction, low central venous saturation and a shorter time to clinical worsening (5.4 months (0.6⁻29.2) vs. 36.5 months (7.4⁻63.4), p = 0.032). Functionally, PAH ECFC had higher proliferative rates compared to control in vitro, although inter-patient variability was high. ECFC proliferation was inversely related to RV end diastolic volume (R² = 0.39, p = 0.018), but not pulmonary vascular resistance. Tube formation-ability was similar among donors. Normal and highly proliferative PAH ECFC were transplanted in pulmonary trunk banded rats. While no effect on hemodynamic measurements was observed, RV vascular density was restored. In conclusion, we found that ECFC outgrowth associates with high clinical severity in PAH, suggesting recruitment. Transplantation of highly proliferative ECFC restored myocardial vascular density in pulmonary trunk banded rats, while RV functional improvements were not observed.

Delayed Microvascular Shear Adaptation in Pulmonary Arterial Hypertension. Role of Platelet Endothelial Cell Adhesion Molecule-1 Cleavage.

RATIONALE: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. OBJECTIVES: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. METHODS: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. MEASUREMENTS AND MAIN RESULTS: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. CONCLUSIONS: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Endothelial damage inhibitor preserves the integrity of venous endothelial cells from patients undergoing coronary bypass surgery.

OBJECTIVES: Despite the success of coronary artery bypass graft (CABG) surgery using autologous saphenous vein grafts (SVGs), nearly 50% of patients experience vein graft disease within 10 years of surgery. One contributing factor to early vein graft disease is endothelial damage during short-term storage of SVGs in inappropriate solutions. Our aim was to evaluate the effects of a novel endothelial damage inhibitor (EDI) on SVGs from patients undergoing elective CABG surgery and on venous endothelial cells (VECs) derived from these SVGs. METHODS: SVGs from 11 patients participating in an ongoing clinical registry (NCT02922088) were included in this study, and incubated with both full electrolyte solution (FES) or EDI for 1 h and then examined histologically. In 8 of 11 patients, VECs were isolated from untreated grafts, incubated with both FES and EDI for 2 h under hypothermic stress conditions and then analysed for activation of an inflammatory phenotype, cell damage and cytotoxicity, as well as endothelial integrity and barrier function. RESULTS: The EDI was superior to FES in protecting the endothelium in SVGs (74 ± 8% versus 56 ± 8%, P < 0.001). Besides confirming that the EDI prevents apoptosis in SVG-derived VECs, we also showed that the EDI temporarily reduces adherens junctions in VECs while protecting focal adhesions compared to FES. CONCLUSIONS: The EDI protects the connectivity and function of the SVG endothelium. Our data suggest that the EDI can preserve focal adhesions in VECs during short-term storage after graft harvesting. This might explain the superiority of the EDI in maintaining most of the endothelium in venous CABG surgery conduits.

Real-time quantification of osteoclastic resorptive activity by electric cell-substrate impedance sensing.

In several diseases, bone resorption by osteoclasts is dysregulated. Thus far, no simple technique for real-time measurement of resorption is available. Here, we introduce an impedimetric bioassay for real-time monitoring of resorption by making use of the electrical insulating properties of the resorbable substrate calcium phosphate. Different chemical stimuli were applied to (pre)osteoclasts cultured on a layer of calcium phosphate in multi-well plates containing electrodes. By this, osteoclast activity can be measured continuously over days, and the effects of stimulating or inhibiting factors can be quantified. When cells were cultured in the presence of an inflammatory factor such as IL-1ß, the resorptive activity started earlier. The measured decline in resistance was higher at culture day 5 than at cultures with M-CSF or M-CSF + RANKL (M-CSF norm. Resistance = 1, M-CSF + RANKL = 0.7, M-CSF + RANKL + IL-1ß = 0.5). However, at day 11, this difference had nearly disappeared. Likewise, bisphosphonates were shown to inhibit osteoclastic activity. Our findings illustrate the importance of real-time monitoring; wherefore, this method has high potential not only for the study of osteoclast resorptive activity in the context of osteoclast function and diseases but also could find application in high-throughput drug-testing studies.

Extracellular Matrix Protein Ratios in the Human Heart and Vessels: How to Distinguish Pathological From Physiological Changes?

Cardiovascular pathology is often accompanied by changes in relative content and/or ratios of structural extracellular matrix (ECM) proteins within the heart and elastic vessels. Three of these proteins, collagen-I, collagen-III, and elastin, make up the bulk of the ECM proteins in these tissues, forming a microenvironment that strongly dictates the tissue biomechanical properties and effectiveness of cardiac and vascular function. In this review, we aim to elucidate how the ratios of collagen-I to collagen-III and elastin to collagen are altered in cardiovascular diseases and the aged individuum. We elaborate on these major cardiovascular ECM proteins in terms of structure, tissue localization, turnover, and physiological function and address how their ratios change in aging, dilated cardiomyopathy, coronary artery disease with myocardial infarction, atrial fibrillation, aortic aneurysms, atherosclerosis, and hypertension. To the end of guiding in vitro modeling approaches, we focus our review on the human heart and aorta, discuss limitations in ECM protein quantification methodology, examine comparability between studies, and highlight potential in vitro applications. In summary, we found collagen-I relative concentration to increase or stay the same in cardiovascular disease, resulting in a tendency for increased collagen-I/collagen-III and decreased elastin/collagen ratios. These ratios were found to fall on a continuous scale with ranges defining distinct pathological states as well as a significant difference between the human heart and aortic ECM protein ratios.

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules. The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

Cellular senescence impairs the reversibility of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) in congenital cardiac shunts can be reversed by hemodynamic unloading (HU) through shunt closure. However, this reversibility potential is lost beyond a certain point in time. The reason why PAH becomes irreversible is unknown. In this study, we used MCT+shunt-induced PAH in rats to identify a dichotomous reversibility response to HU, similar to the human situation. We compared vascular profiles of reversible and irreversible PAH using RNA sequencing. Cumulatively, we report that loss of reversibility is associated with a switch from a proliferative to a senescent vascular phenotype and confirmed markers of senescence in human PAH-CHD tissue. In vitro, we showed that human pulmonary endothelial cells of patients with PAH are more vulnerable to senescence than controls in response to shear stress and confirmed that the senolytic ABT263 induces apoptosis in senescent, but not in normal, endothelial cells. To support the concept that vascular cell senescence is causal to the irreversible nature of end-stage PAH, we targeted senescence using ABT263 and induced reversal of the hemodynamic and structural changes associated with severe PAH refractory to HU. The factors that drive the transition from a reversible to irreversible pulmonary vascular phenotype could also explain the irreversible nature of other PAH etiologies and provide new leads for pharmacological reversal of end-stage PAH.