Plexiform Lesions Research Articles

Recruitment of CCR5-positive T cells in pulmonary vascular lesions in idiopathic- and connective tissue disease associated- pulmonary arterial hypertension

Abstract Background C-C chemokine receptor type 5 (CCR5) is a chemokine receptor predominantly expressed on leukocytes including T cells, macrophages, and dendritic cells, and is involved in the process by which T cells are attracted to specific tissues and organs. Although CCR5 has been shown to be expressed on macrophages and pulmonary artery smooth muscle cells in lung samples from human idiopathic pulmonary arterial hypertension (PAH) and some animal models, its expression on T cells in human PAH lungs has not been investigated. Purpose To investigate the immunohistochemical expression of CCR5 on T cells infiltrating in pulmonary vascular lesions of human PAH tissues, particularly from patients with idiopathic PAH and PAH associated with connective tissue disease (CTD-PAH), where inflammation has been implicated in the pathogenesis. Methods Subjects included 25 postmortem cases of idiopathic PAH (14 cases, a mean age of 34 ± 10 years, 8 female cases) and CTD-PAH (11 cases, 48 ± 13 years, all female). We performed immunohistochemistry using antibodies against CCR5 and CD3 (for T cells) on formalin-fixed paraffin-embedded sections. To semi-quantify, we selected the three small muscular pulmonary arteries (80-150 µm in external diameter) with the strongest CCR5-positive cell infiltration in each case, counted the number of positive cells, and the average were shown as the number of cells per vessel. Results In all cases, regardless of whether IPAH or CTD-PAH, a subset of infiltrating inflammatory cells into or accumulating around vessels was CCR5-positive. Specifically, CCR5-positive inflammatory cells were found in remodelled pulmonary arteries, such as plexiform lesions and constrictive lesions with intimal thickening and/or medial hypertrophy, as well as perivascular space. The average number of CCR5-positive cells per small muscular pulmonary artery was 8.1/vessel. Where pulmonary venous occlusive lesions were seen, CCR5-positive cells were also present in and around them. The majority of CCR5-positive inflammatory cells appeared to be T cells, based on morphology and CD3 immunopositivity in the serial sections. Conclusion(s) Activated T cells showing CCR5 immunopositivity were recruited to remodelled pulmonary vascular lesions in both idiopathic PAH and CTD-PAH. This implicates CCR5-positive T cells as a common key player and that CCR5 signalling is involved in the pathogenesis of PAH in concert with other inflammatory cells, vascular endothelial cells, and pulmonary artery smooth muscle cells.

Case of Bone Morphogenetic Protein Receptor Type 2 (BMPR2) Mutation Complicated by Pulmonary Hypertension Encountered at Autopsy

Abstract Introduction/Objective Bone morphogenetic protein receptor type 2 is a protein encoded by the BMPR2 gene which regulates cell division and apoptosis. BMPR2 mutations result in monoclonal plexiform lesions of proliferating endothelial cells in pulmonary arterioles, which leads to elevated pulmonary artery pressures, right ventricular failure, and death. Here, we present the case of a 12-year-old male with a known history of pulmonary hypertension secondary to BMPR2 mutation referred for medical autopsy after suffering cardiac arrest and passing away. Methods/Case Report This 12-year-old male was diagnosed with pulmonary hypertension in December 2022 after suffering a syncopal episode while running at recess. Genetic testing was obtained, and he was found to have a BMPR2 mutation. His father was tested and also harbored this mutation, but did not clinically have pulmonary hypertension. The decedent was initiated on ambrisentan, tadalafil, and spironolactone, but continued to present to the Emergency Department periodically for syncopal episodes. In April 2024, he presented to the Medical University of South Carolina in cardiac arrest which began while playing outside. A complete autopsy was performed, including gross and microscopic examination of tissues and postmortem vitreous electrolyte analysis. Gross examination revealed the decedent’s heart was enlarged to 350 grams (normal mean for age = 181 grams). Both cardiac ventricles were dilated and the right ventricle was markedly hypertrophic with a thickness of 1.2 cm. Microscopic sections are pending but will presumptively show significant cardiac myocyte hypertrophy and pulmonary arteriole thickening. Cause of death is pending certification but is presumed to be sudden cardiac death resulting from BMPR2 mutation- related pulmonary hypertension. Results (if a Case Study enter NA) NA Conclusion This case highlights a common genetic presentation of pulmonary arterial hypertension, with approximately 80% of familial cases of pulmonary arterial hypertension harboring BMPR2 mutations. Further research should be done to investigate treatment options and management of this genetic presentation.

Current Overview of the Biology and Pharmacology in Sugen/Hypoxia-Induced Pulmonary Hypertension in Rats.

The Sugen 5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension (PAH) demonstrates most of the distinguishing features of PAH in humans, including increased wall thickness and obstruction of the small pulmonary arteries along with plexiform lesion formation. Recently, significant advancement has been made describing the epidemiology, genomics, biochemistry, physiology, and pharmacology in Su/Hx challenge in rats. For example, there are differences in the overall reactivity to Su/Hx challenge in different rat strains and only female rats respond to estrogen treatments. These conditions are also encountered in human subjects with PAH. Also, there is a good translation in both the biochemical and metabolic pathways in the pulmonary vasculature and right heart between Su/Hx rats and humans, particularly during the transition from the adaptive to the nonadaptive phase of right heart failure. Noninvasive techniques such as echocardiography and magnetic resonance imaging have recently been used to evaluate the progression of the pulmonary vascular and cardiac hemodynamics, which are important parameters to monitor the efficacy of drug treatment over time. From a pharmacological perspective, most of the compounds approved clinically for the treatment of PAH are efficacious in Su/Hx rats. Several compounds that show efficacy in Su/Hx rats have advanced into phase II/phase III studies in humans with positive results. Results from these drug trials, if successful, will provide additional treatment options for patients with PAH and will also further validate the excellent translation that currently exists between Su/Hx rats and the human PAH condition.

Multiparametric whole-body MRI of patients with neurofibromatosis type I: spectrum of imaging findings.

Neurofibromatosis (NF) type I is a neuroectodermal and mesodermal dysplasia caused by a mutation of the neurofibromin tumor suppressor gene. Phenotypic features of NF1 vary, and patients develop benign peripheral nerve sheath tumors and malignant neoplasms, such as malignant peripheral nerve sheath tumor, malignant melanoma, and astrocytoma. Multiparametric whole-body MR imaging (WBMRI) plays a critical role in disease surveillance. Multiparametric MRI, typically used in prostate imaging, is a general term for a technique that includes multiple sequences, i.e. anatomic, diffusion, and Dixon-based pre- and post-contrast imaging. This article discusses the value of multiparametric WBMRI and illustrates the spectrum of whole-body lesions of NF1 in a single imaging setting. Examples of lesions include those in the skin (tumors and axillary freckling), soft tissues (benign and malignant peripheral nerve sheath tumors, visceral plexiform, and diffuse lesions), bone and joints (nutrient nerve lesions, non-ossifying fibromas, intra-articular neurofibroma, etc.), spine (acute-angled scoliosis, dural ectasia, intraspinal tumors, etc.), and brain/skull (optic nerve glioma, choroid plexus xanthogranuloma, sphenoid wing dysplasia, cerebral hamartomas, etc.). After reading this article, the reader will gain knowledge of the variety of lesions encountered with NF1 and their WBMRI appearances. Timely identification of such lesions can aid in accurate diagnosis and appropriate patient management.

Genetic variants of the BMPR2 gene and their contribution to the development of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive increase in pressure in the pulmonary arteries. This leads to vascular remodeling and, eventually, right heart failure. Variants in the BMPR2 gene, which encodes the bone morphogenetic protein receptor type 2, are the most common genetic cause of hereditary and idiopathic PAH. These variants disrupt the transforming growth factor-beta (TGF-β) signaling pathway, triggering abnormal proliferation of pulmonary artery smooth muscle cells and apoptosis of endothelial cells. This results in complex vascular remodeling, characterized by thickening of the vascular walls, formation of plexiform lesions, and in situ thrombosis. Endothelial dysfunction also contributes to an imbalance between vasoconstrictors and vasodilators, exacerbating pulmonary hypertension. While current therapies aim to restore this balance, they have a limited impact on the underlying vascular remodeling. Emerging strategies seek to restore the functionality of BMPR2 variants or enhance their signaling through agonists and read-through molecules. Additionally, a higher penetrance of the disease has been observed in women carrying BMPR2 variants, suggesting an interaction with sex hormone metabolism. The research aims to understand the role of BMPR2 gene variants in the development of PAH and to explore emerging strategies to restore the functionality of these variants or enhance their signaling.

STAT3 phosphorylation at Tyr705 affects DRP1 (dynamin-related protein 1) controlled-mitochondrial fission during the development of apoptotic-resistance in pulmonary arterial endothelial cells

BackgroundThe apoptosis-resistant pulmonary arterial endothelial cells (PAECs) are known to be major players in the pulmonary remodeling of pulmonary arterial hypertension (PAH) and exhibit an abnormal metabolic profile with mitochondrial dysfunction. Mitochondrial fission has been shown to regulate the apoptosis of several cell types, but this is largely unexplored in the PAECs.ObjectiveThe roles of mitochondrial fission control by Dynamin related protein-1 (DRP1) in the development of PAECs apoptosis suppression were investigated in present study and the potential mechanisms behind this were furtherly explored.MethodsThe mitochondrial morphology was investigated in PAECs from PAH rats with the pulmonary plexiform lesions, and the relations of it with DRP1 expression and apoptosis were furtherly identified in apoptosis-resistant PAECs induced by hypoxia. PAECs were isolated from rats with severe PAH and from normal subjects, the apoptotic-resistant PAECs were induced by hypoxia. DRP1 gene knockdown was achieved via DRP1-siRNA, DRP1 and STAT3 phosphorylation were blocked using its inhibitors, respectively. Apoptosis was analyzed by flow cytometry, and mitochondrial morphology was investigated by transmission electron microscope and confocal microscopy.ResultsThe PAECs isolated from PAH rats with the pulmonary plexiform-like lesions and displayed lower apoptotic rate with increased DRP1 expression and mitochondrial fragmentation. In addition, similar observations were achieved in apoptosis-resistant PAECs induced by hypoxia. Targeting DRP1 using siRNA and pharmacologic blockade prevented the mitochondrial fission and subsequent apoptotic resistance in PAECs under hypoxia. Mechanistically, STAT3 phosphorylation at Tyr705 was shown to be activated in both PAH and hypoxia-treated PAECs, leading to the regulation of DRP1 expression. Of importance, targeting STAT3Tyr705 phosphorylation prevented DRP1 disruption on apoptosis in PAECs under hypoxia.ConclusionsThese data indicated that STAT3 phosphorylation at Tyr705 impacted DRP1-controlled mitochondrial fission during the development of apoptosis-resistance in PAECs, suggesting mitochondrial dynamics may represent a therapeutic target for PAH.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

Digital Spatial Profiling Identifies Distinct Molecular Signatures of Vascular Lesions in Pulmonary Arterial Hypertension.

Rationale: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by extensive pulmonary vascular remodeling caused by plexiform and obliterative lesions, media hypertrophy, inflammatory cell infiltration, and alterations of the adventitia. Objective: We sought to test the hypothesis that microscopic IPAH vascular lesions express unique molecular profiles, which collectively are different from control pulmonary arteries. Methods: We used digital spatial transcriptomics to profile the genomewide differential transcriptomic signature of key pathological lesions (plexiform, obliterative, intima+media hypertrophy, and adventitia) in IPAH lungs (n = 11) and compared these data with the intima+media hypertrophy and adventitia of control pulmonary artery (n = 5). Measurements and Main Results: We detected 8,273 transcripts in the IPAH lesions and control lung pulmonary arteries. Plexiform lesions and IPAH adventitia exhibited the greatest number of differentially expressed genes when compared with intima+media hypertrophy and obliterative lesions. Plexiform lesions in IPAH showed enrichment for 1) genes associated with transforming growth factor β signaling and 2) mutated genes affecting the extracellular matrix and endothelial-mesenchymal transformation. Plexiform lesions and IPAH adventitia showed upregulation of genes involved in immune and IFN signaling, coagulation, and complement pathways. Cellular deconvolution indicated variability in the number of vascular and inflammatory cells between IPAH lesions, which underlies the differential transcript profiling. Conclusions: IPAH lesions express unique molecular transcript profiles enriched for pathways involving pathogenetic pathways, including genetic disease drivers, innate and acquired immunity, hypoxia sensing, and angiogenesis signaling. These data provide a rich molecular-structural framework in IPAH vascular lesions that inform novel biomarkers and therapeutic targets in this highly morbid disease.

The NF1+/- Immune Microenvironment: Dueling Roles in Neurofibroma Development and Malignant Transformation.

Neurofibromatosis type 1 (NF1) is a common genetic disorder resulting in the development of both benign and malignant tumors of the peripheral nervous system. NF1 is caused by germline pathogenic variants or deletions of the NF1 tumor suppressor gene, which encodes the protein neurofibromin that functions as negative regulator of p21 RAS. Loss of NF1 heterozygosity in Schwann cells (SCs), the cells of origin for these nerve sheath-derived tumors, leads to the formation of plexiform neurofibromas (PNF)-benign yet complex neoplasms involving multiple nerve fascicles and comprised of a myriad of infiltrating stromal and immune cells. PNF development and progression are shaped by dynamic interactions between SCs and immune cells, including mast cells, macrophages, and T cells. In this review, we explore the current state of the field and critical knowledge gaps regarding the role of NF1(Nf1) haploinsufficiency on immune cell function, as well as the putative impact of Schwann cell lineage states on immune cell recruitment and function within the tumor field. Furthermore, we review emerging evidence suggesting a dueling role of Nf1+/- immune cells along the neurofibroma to MPNST continuum, on one hand propitiating PNF initiation, while on the other, potentially impeding the malignant transformation of plexiform and atypical neurofibroma precursor lesions. Finally, we underscore the potential implications of these discoveries and advocate for further research directed at illuminating the contributions of various immune cells subsets in discrete stages of tumor initiation, progression, and malignant transformation to facilitate the discovery and translation of innovative diagnostic and therapeutic approaches to transform risk-adapted care.

Unilateral Lung Removal in Combination with Monocrotaline or SU5416 in Rodents: A Reliable Model to Mimic the Pathology of the Human Pulmonary Hypertension.

Pulmonary hypertension (PH) is a chronic and progressive disorder characterized by elevated mean pulmonary arterial pressure, pulmonary vascular remodeling, and the development of concentric laminar intimal fibrosis with plexiform lesions. While rodent models have been developed to study PH, they have certain deficiencies and do not entirely replicate the human disease due to the heterogeneity of PH pathology. Therefore, combined models are necessary to study PH. Recent studies have shown that altered pulmonary blood flow is a significant trigger in the development of vascular remodeling and neointimal lesions. One of the most promising rodent models for increased pulmonary flow is the combination of unilateral left pneumonectomy with a "second hit" of monocrotaline (MCT) or SU5416. The removal of one lung in this model forces blood to circulate only in the other lung and induces increased and turbulent pulmonary blood flow. This increased vascular flow leads to progressive remodeling and occlusion of small pulmonary arteries. The second hit by MCT or SU5416 leads to endothelial cell dysfunction, resulting in severe PH and the development of plexiform arteriopathy.

Clinical-radiological-pathological correlation in pulmonary arterial hypertension.

Pulmonary hypertension (PH) is defined by the presence of a mean pulmonary arterial pressure >20 mmHg. Current guidelines describe five groups of PH with shared pathophysiological and clinical features. In this paper, the first of a series covering all five PH classification groups, the clinical, radiological and pathological features of pulmonary arterial hypertension (PAH) will be reviewed. PAH may develop in the presence of associated medical conditions or a family history, following exposure to certain medications or drugs, or may be idiopathic in nature. Although all forms of PAH share common histopathological features, the presence of certain pulmonary arterial abnormalities, such as plexiform lesions, and extent of co-existing pulmonary venous involvement differs between the different subgroups. Radiological investigations are key to diagnosing the correct form of PH and a systematic approach to interpretation, especially of computed tomography, is essential.

Abstract 11703: Proteomic Analysis for the Formation of the Plexiform Lesions of a Rat Pulmonary Arterial Hypertension Model

Introduction: Although plexiform lesions (PLs) are the hallmark of the severe pulmonary arterial hypertension, the mechanism of this lesion formation has not been fully elucidated. In this study, to explore the pathways associated PLs formation, we examined the proteome analysis using vascular tissue before and after the formation of PLs obtained by laser capture microdissection (LCM). Methods: 7 weeks old rats were injected Sugen 5416 subcutaneously, followed by exposed to hypoxia for 3 weeks. In addition, the rats were kept in room air for 5 weeks (8-week model) or 10 weeks (13-week model) thereafter. We obtained the tissue of vessels with medial hypertrophy from 8-week models (n=5) and PLs from 13-week models (n=5) by LCM. Those micro-dissected samples were then subject to a qualitative and then quantitative proteomics approach. Differentially expressed proteins (DEPs) were defined by abundance in mass spectrometry (MS) (P-value < 0.05 and |log2 (fold change)| > 0.5 or proteins deteced only in lesions extracted from one model). In addition, we investigated enhanced pathway by gene ontology (GO) analysis of the identified DEPs in each of the 8-week and 13-week models. Results: We identified 718 uniquely expressed proteins including 57 DEPs (31 in 8-week model versus 26 in 13-week model) by using LCM-MS. GO analysis revealed that the actin cytoskeleton organization, actin filament-based process, and actomyosin structure organization processes were enhanced in 8-week model. On the other hand, the innate immune system, G2/M check points, and cellular response to reactive oxygen species were enhanced in 13-week model. Conclusion: Pulmonary vascular lesions of 8-week and PLs of 13-week Sugen hypoxia model evaluated by proteomics using samples obtained by LCM methods showed differential protein expression. Analysis of these DEPs may contribute to elucidate the mechanisms of PLs formation.

Pharmacology and Rationale for Seralutinib in the Treatment of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a complex disorder characterized by vascular remodeling and a consequent increase in pulmonary vascular resistance. The histologic hallmarks of PAH include plexiform and neointimal lesions of the pulmonary arterioles, which are composed of dysregulated, apoptosis-resistant endothelial cells and myofibroblasts. Platelet-derived growth factor receptors (PDGFR) α and β, colony stimulating factor 1 receptor (CSF1R), and mast/stem cell growth factor receptor kit (c-KIT) are closely related kinases that have been implicated in PAH progression. In addition, emerging data indicate significant crosstalk between PDGF signaling and the bone morphogenetic protein receptor type 2 (BMPR2)/transforming growth factor β (TGFβ) receptor axis. This review will discuss the importance of the PDGFR-CSF1R-c-KIT signaling network in PAH pathogenesis, present evidence that the inhibition of all three nodes in this kinase network is a potential therapeutic approach for PAH, and highlight the therapeutic potential of seralutinib, currently in development for PAH, which targets these pathways.

Giant Plexiform Neurofibroma of the Thigh: A Case Report

Neurofibromas are known to manifest most frequently as localized lesions, less frequently as a diffuse form, and rarely as a plexiform variety We report the clinical and imaging features of a young male patient with biopsy proven plexiform neurofibroma (PNF). A 23 years old male, followed for neurofibromatosis type 1, plexiform lesions of the left lower limb progressively increasing in volume with scrotal extension. The MRI showed thickening of the cutaneous and subcutaneous soft tissues on the medial and posterior aspect of the left thigh, extending to the perineo-scrotal region. The role of imaging is important for a variety of reasons, including delineating the extent of involvement and effect on adjacent structures, exposing associated anomalies and last but not least, for predicting possible malignant transformation. MRI is the reference standard modality for evaluating neural tissues and also for delineating the parent nerve in cases of tumors of neural origin. Therapy of plexiform neurofibromas is usually surgical, aiming at resecting deforming masses and cancerous tissue when malignant transformation occurs.

HIV- RELATED PULMONARY ARTERIAL HYPERTENSION: A CASE REPORT

The use of highly active antiretroviral therapy (HAART) has increased the life expectancy of HIV-infected patients. With prolonged survival and improved control of infectious susceptibility, vascular complications have emerged as a signicant source of morbidity and mortality in HIV-infected patients [1]. HIV-associated pulmonary arterial hypertension (HIV-PAH) is an important lung disease in HIV-infected persons who live longer with antiretrovirals. HIV-PAH may be detected via chest radiographs, CT scans, or electrocardiograms, but Doppler echocardiography is the most useful screening test to identify candidates for right heart catheterization. Because the survival for HIV-infected individuals with PAH with advanced symptoms (New York Heart Association, NYHA class III-IV) is worse compared with less symptomatic individuals (NYHA class I-II) (2), identication of asymptomatic individuals is of critical importance. Histologically, THE lesions in HIV-infected patients with PAH include concentric laminar intimal brosis, medial hypertrophy, recanalized thrombi, and plexiform lesions (3). According to a more recent study by Sitbon et al [4] in 2008, the prevalence has remained at 0.5% even in the modern era of HIV therapy, suggesting that HAART has not made a dramatic impact on the prevention of HIV-PAH. We present a case of HIVPAH to highlight the importance of the non-infectious pulmonary complications associated with HIV disease.

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

Second International Pulmonary Hypertension/Heart Failure Symposium—Structural heart disease, right ventricular dysfunction, and stem cell therapy: The European Pediatric Pulmonary Vascular Disease Network

Abstract 15659: Development of Novel EMAP-II Neutralizing Antibody Therapy for HIV-Associated Pulmonary Hypertension via Abrogation of HIV-Nef Induced Pulmonary Endothelial Damage

Rationale: People living with HIV experience increased rates of cardiovascular co-morbidities including pulmonary hypertension (600-fold higher prevalence) despite immediate prescription of combined antiviral therapy (ART) upon HIV diagnosis. Previously, we reported that the virally encoded HIV-Nef protein persists in extracellular vesicles (EV) isolated from bronchoalveolar lavage fluid of aviremic HIV patients on ART. Interestingly, Nef-positivity correlated with increased EMAPII levels, a cellular protein which when released extracellularly specifically amplifies endothelial apoptosis. We hypothesize that Nef-EMAPII signaling axis induces endothelial apoptosis and senescence, which could give rise to apoptosis-senescence-resistant, plexiform lesion-forming endothelial cells. Methods and Results: Co-culture with Nef EV producing SupT1 T-cells induces endothelial senescence quantified using senescence associated marker-p16INK4a (p<0.001) which corresponds to increased EMAPII surface expression. Nef induced endothelial senescence as quantified by β-Galactosidase activity was blocked with EMAPII neutralizing antibody (Fig). Transgenic mice with endothelium specific Nef expression had elevated EMAP II surface expression. Two-dose injection of EMAPII neutralizing antibodies (1 mg/mouse, sc) abrogates apoptosis induction as determined by cleaved caspase 3+ pulmonary endothelial cells (p<0.001). Furthermore, Nef Tg mice derived pulmonary endothelial cells had reduced colony formation (p<0.001) which is being tested with EMAPII neutralization as a therapeutic agent. Conclusion: Nef induced apoptosis and cellular senescence can be targeted with EMAP II neutralizing antibodies both in vitro and in vivo . These data suggest that A) Nef can give rise to apoptosis-senescence-resistant plexiform lesion forming endothelial cells and B) neutralizing EMAPII antibodies can be employed to block progression of PAH.

A Protocol for Fabrication and on-Chip Cell Culture to Recreate PAH-Afflicted Pulmonary Artery on a Microfluidic Device.

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disease that affects people of all ethnic origins and age groups including newborns. In PAH, pulmonary arteries and arterioles undergo a series of pathological changes including remodeling of the entire pulmonary vasculatures and extracellular matrices, mis-localized growth of pulmonary arterial cells, and development of glomeruloid-like lesions called plexiform lesions. Traditionally, various animal and cellular models have been used to understand PAH pathophysiology, investigate sex-disparity in PAH and monitor therapeutic efficacy of PAH medications. However, traditional models can only partially capture various pathological features of PAH, and they are not adaptable to combinatorial study design for deciphering intricately intertwined complex cellular processes implicated in PAH pathogenesis. While many microfluidic chip-based models are currently available for major diseases, no such disease-on-a-device model is available for PAH, an under investigated disease. In the absence of any chip-based models of PAH, we recently proposed a five-channel polydimethylsiloxane (PDMS)-based microfluidic device that can emulate major pathological features of PAH. However, our proposed model can make a bigger impact on the PAH field only when the larger scientific community engaged in PAH research can fabricate the device and develop the model in their laboratory settings. With this goal in mind, in this study, we have described the detailed methodologies for fabrication and development of the PAH chip model including a thorough explanation of scientific principles for various steps for chip fabrication, a detailed list of reagents, tools and equipment along with their source and catalogue numbers, description of laboratory setup, and cautionary notes. Finally, we explained the methodologies for on-chip cell seeding and application of this model for studying PAH pathophysiology. We believe investigators with little or no training in microfluidic chip fabrication can fabricate this eminently novel PAH-on-a-chip model. As such, this study will have a far-reaching impact on understanding PAH pathophysiology, unravelling the biological mystery associated with sexual dimorphism in PAH, and developing PAH therapy based on patient sex and age.

Immunohistochemical profile of the pulmonary vasculature in subjects with cirrhosis and histopathologic evidence of pulmonary vascular disease: An autopsy study

Portopulmonary hypertension (POPH) is a complication of cirrhosis that results in right ventricular failure and death. The objective of this autopsy investigation was to compare pulmonary arterial receptors in subjects with cirrhosis and histopathologic evidence of pulmonary vascular disease (PVD) and control group subjects with cirrhosis lacking evidence of PVD. Autopsy records of 824 subjects with cirrhosis were reviewed to identify pulmonary arterial vasculopathy. Lung sections from paraffin embedded blocks were immunostained for endothelin A (ET-A), endothelin B (ET- B), estrogen α (ER-α), estrogen β (ER-β), and vascular endothelial growth factor (VEGF). Subjects with cirrhosis and histopathologic evidence of PVD included 27 individuals with intimal hyperplasia (93%), medial hypertrophy (96%), and plexiform lesions (78%). Immunohistochemical staining for ET-A revealed positive reactivity in 40% of the group with cirrhosis and histopathologic evidence of PVD and 13% of the control group (NS). ET-B reactivity in the pulmonary endothelium and smooth muscle was identified in all subjects with cirrhosis and histopathologic evidence of PVD and control group. VEGF reactivity was identified in the endothelium in all subjects with cirrhosis and histopathologic evidence of PVD compared with 33% of the control group (p = 0.0002). ER-β reactivity was observed in four subjects (26.6%) with cirrhosis and histopathologic evidence of PVD while none in the control group (NS). Cirrhosis and histopathologic evidence of PVD was found in 3.3% of autopsies with the pulmonary vasculature immunohistochemical profile demonstrating endothelial and smooth muscle reactivity for endothelin, VEGF and ER-β.

Transgelin exacerbates pulmonary artery smooth muscle cell dysfunction in shunt-related pulmonary arterial hypertension.

Orchestrating the transition from reversible medial hypertrophy to irreversible plexiform lesions is crucial for pulmonary arterial hypertension related to congenital heart disease (CHD-PAH). Transgelin is an actin-binding protein that modulates pulmonary arterial smooth muscle cell (PASMC) dysfunction. In this study, we aimed to probe the molecular mechanism and biological function of transgelin in the pathogenesis of CHD-PAH. Transgelin expression was detected in lung tissues from both CHD-PAH patients and monocrotaline (MCT)-plus aortocaval (AV)-induced PAH rats by immunohistochemistry. In vitro, the effects of transgelin on the proliferation, migration, and apoptosis of human PASMCs (HPASMCs) were evaluated by the cell count and EdU assays, transwell migration assay, and TUNEL assay, respectively. And the effect of transgelin on the expression of HPASMC phenotype markers was assessed by the immunoblotting assay. (i) Compared with the normal control group (n=12), transgelin expression was significantly overexpressed in the pulmonary arterioles of the reversible (n=15) and irreversible CHD-PAH group (n=4) (reversible group vs. control group: 18.2±5.1 vs. 13.6±2.6%, P<0.05; irreversible group vs. control group: 29.9±4.7 vs. 13.6±2.6%, P<0.001; irreversible group vs. reversible group: 29.9±4.7 vs. 18.2±5.1, P<0.001). This result was further confirmed in MCT-AV-induced PAH rats. Besides, the transgelin expression level was positively correlated with the pathological grading of pulmonary arteries in CHD-PAH patients (r=0.48, P=0.03, n=19). (ii) Compared with the normal control group (n=12), TGF-β1 expression was notably overexpressed in the pulmonary arterioles of the reversible (n=15) and irreversible CHD-PAH group (n=4) (reversible group vs. control group: 14.8±4.4 vs. 6.0±2.5%, P<0.001; irreversible group vs. control group: 20.1±4.4 vs. 6.0±2.5%, P<0.001; irreversible group vs. reversible group: 20.1±4.4 vs. 14.8±4.4, P<0.01). The progression-dependent correlation between TGF-β1 and transgelin was demonstrated in CHD-PAH patients (r=0.48, P=0.04, n=19) and MCT-AV-induced PAH rats, which was further confirmed at sub-cellular levels. (iii) Knockdown of transgelin diminished proliferation, migration, apoptosis resistance, and phenotypic transformation of HPASMCs through repressing the TGF-β1 signalling pathway. On the contrary, transgelin overexpression resulted in the opposite effects. These results indicate that transgelin may be an indicator of CHD-PAH development via boosting HPASMC dysfunction through positive regulation of the TGF-β1 signalling pathway, as well as a potential therapeutic target for the treatment of CHD-PAH.