Notch3 deletion regulates HIV-1 gene expression and systemic inflammation to ameliorate chronic kidney disease.

Antiretroviral therapy (ART) has profoundly decreased HIV-1 associated morbidity. However, despite ART, immune cells remain latently infected and slowly release viral proteins, leading to chronic inflammation and HIV associated comorbidities. Thus, new strategies are needed to reduce the inflammatory effects of HIV-1. In previous studies we found that gamma secretase inhibitor (GSIXX) ameliorated renal lesions of HIV-Tg26 mice carrying replication defective HIV-1 PNL4-3 by inhibiting Notch activation. Since gamma secretase inhibition is not a safe strategy in humans, here we examined the specific role of the Notch3 pathway in the pathogenesis of the renal lesions and outcome of HIV-Tg26 mice. We found that Notch3 is activated in podocytes and other renal cells in HIV-Tg26 mice and human biopsies with HIV-1 associated Nephropathy (HIVAN). Knockdown of Notch3 in HIV-Tg26 mice revealed a marked reduction in the mortality rate, improvement in renal injury and function. RNA sequencing and immunolabeling data revealed that Notch3 deletion drastically reduced infiltrating renal macrophages in HIV-Tg-N3KO mice in association with renal reduction of HIV-nef mRNA expression levels. In fact, bone marrow derived macrophages from HIV-Tg26 mice showed a significant activation of Notch3 signaling. Further, systemic levels of TNF-alpha and MCP-1 and other inflammatory chemokines and cytokines were reduced in Tg-N3KO mice as compared to HIV-Tg26 mice and this translated to a marked reduction of HIV-induced skin lesions. Taken together, these studies strongly point to a dual inhibitory/therapeutic effect of Notch3 inhibition on HIV-induced systemic, skin and renal lesions independently of ART.

HIV and Drug Use: A Tale of Synergy in Pulmonary Vascular Disease Development.

Over the past two decades, with the advent and adoption of highly active anti-retroviral therapy, HIV-1 infection, a once fatal and acute illness, has transformed into a chronic disease with people living with HIV (PWH) experiencing increased rates of cardio-pulmonary vascular diseases including life-threatening pulmonary hypertension. Moreover, the chronic consequences of tobacco, alcohol, and drug use are increasingly seen in older PWH. Drug use, specifically, can have pathologic effects on the cardiovascular health of these individuals. The "double hit" of drug use and HIV may increase the risk of HIV-associated pulmonary arterial hypertension (HIV-PAH) and potentiate right heart failure in this population. This article explores the epidemiology and pathophysiology of PAH associated with HIV and recreational drug use and describes the proposed mechanisms by which HIV and drug use, together, can cause pulmonary vascular remodeling and cardiopulmonary hemodynamic compromise. In addition to detailing the proposed cellular and signaling pathways involved in the development of PAH, this article proposes areas ripe for future research, including the influence of gut dysbiosis and cellular senescence on the pathobiology of HIV-PAH. © 2023 American Physiological Society. Compr Physiol 13:4659-4683, 2023.

Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19.

SARS-CoV-2, the causative agent of COVID-19 disease, has resulted in the death of millions worldwide since the beginning of the pandemic in December 2019. While much progress has been made to understand acute manifestations of SARS-CoV-2 infection, less is known about post-acute sequelae of COVID-19 (PASC). We investigated the levels of both Spike protein (Spike) and viral RNA circulating in patients hospitalized with acute COVID-19 and in patients with and without PASC. We found that Spike and viral RNA were more likely to be present in patients with PASC. Among these patients, 30% were positive for both Spike and viral RNA; whereas, none of the individuals without PASC were positive for both. The levels of Spike and/or viral RNA in the PASC+ve patients were found to be increased or remained the same as in the acute phase; whereas, in the PASC-ve group, these viral components decreased or were totally absent. Additionally, this is the first report to show that part of the circulating Spike is linked to extracellular vesicles without any presence of viral RNA in these vesicles. In conclusion, our findings suggest that Spike and/or viral RNA fragments persist in the recovered COVID-19 patients with PASC up to 1 year or longer after acute SARS-CoV-2 infection.

Potential long-term effects of SARS-CoV-2 infection on the pulmonary vasculature: Multilayered cross-talks in the setting of coinfections and comorbidities.

The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its sublineages pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available therapies. COVID-19, although targeting primarily the respiratory system, is also now well established that later affects every organ in the body. Most importantly, despite the available therapy and vaccine-elicited protection, the long-term consequences of viral infection in breakthrough and asymptomatic individuals are areas of concern. In the past two years, investigators accumulated evidence on how the virus triggers our immune system and the molecular signals involved in the cross-talk between immune cells and structural cells in the pulmonary vasculature to drive pathological lung complications such as endothelial dysfunction and thrombosis. In the review, we emphasize recent updates on the pathophysiological inflammatory and immune responses associated with SARS-CoV-2 infection and their potential long-term consequences that may consequently lead to the development of pulmonary vascular diseases.

Exploring extracellular vesicles as mediators of clinical disease and vehicles for viral therapeutics: Insights from the COVID-19 pandemic.

The COVID-19 pandemic has challenged researchers to rapidly understand the capabilities of the SARS-CoV-2 virus and investigate potential therapeutics for SARS-CoV-2 infection. COVID-19 has been associated with devastating lung and cardiac injury, profound inflammation, and a heightened coagulopathic state, which may, in part, be driven by cellular crosstalk facilitated by extracellular vesicles (EVs). In recent years, EVs have emerged as important biomarkers of disease, and while extracellular vesicles may contribute to the spread of COVID-19 infection from one cell to the next, they also may be engineered to play a protective or therapeutic role as decoys or "delivery drivers" for therapeutic agents. This review explores these roles and areas for future study.

Extracellular vesicle-mediated endothelial apoptosis and EV-associated proteins correlate with COVID-19 disease severity.

Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue-remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets.

Impact of human immunodeficiency virus on pulmonary vascular disease.

With the advent of anti-retroviral therapy, non-AIDS-related comorbidities have increased in people living with HIV. Among these comorbidities, pulmonary hypertension (PH) is one of the most common causes of morbidity and mortality. Although chronic HIV-1 infection is independently associated with the development of pulmonary arterial hypertension, PH in people living with HIV may also be the outcome of various co-morbidities commonly observed in these individuals including chronic obstructive pulmonary disease, left heart disease and co-infections. In addition, the association of these co-morbidities and other risk factors, such as illicit drug use, can exacerbate the development of pulmonary vascular disease. This review will focus on these complex interactions contributing to PH development and exacerbation in HIV patients. We also examine the interactions of HIV proteins, including Nef, Tat, and gp120 in the pulmonary vasculature and how these proteins alter the endothelial and smooth muscle function by transforming them into susceptible PH phenotype. The review also discusses the available infectious and non-infectious animal models to study HIV-associated PAH, highlighting the advantages and disadvantages of each model, along with their ability to mimic the clinical manifestations of HIV-PAH.

Extracellular Vesicle TGF-ß1 Is Linked to Cardiopulmonary Dysfunction in Human Immunodeficiency Virus.

Extracellular vesicles (EVs) have emerged as important mediators in cell-cell communication; however, their relevance in pulmonary hypertension (PH) secondary to human immunodeficiency virus (HIV) infection is yet to be explored. Considering that circulating monocytes are the source of the increased number of perivascular macrophages surrounding the remodeled vessels in PH, this study aimed to identify the role of circulating small EVs and EVs released by HIV-infected human monocyte-derived macrophages in the development of PH. We report significantly higher numbers of plasma-derived EVs carrying higher levels of TGF-ß1 (transforming growth factor-ß1) in HIV-positive individuals with PH compared with individuals without PH. Importantly, levels of these TGF-ß1-loaded, plasma-derived EVs correlated with pulmonary arterial systolic pressures and CD4 counts but did not correlate with the Dl CO or viral load. Correspondingly, enhanced TGF-ß1-dependent pulmonary endothelial injury and smooth muscle hyperplasia were observed. HIV-1 infection of monocyte-derived macrophages in the presence of cocaine resulted in an increased number of TGF-ß1-high EVs, and intravenous injection of these EVs in rats led to increased right ventricle systolic pressure accompanied by myocardial injury and increased levels of serum ET-1 (endothelin-1), TNF-α, and cardiac troponin-I. Conversely, pretreatment of rats with TGF-ß receptor 1 inhibitor prevented these EV-mediated changes. Findings define the ability of macrophage-derived small EVs to cause pulmonary vascular modeling and PH via modulation of TGF-ß signaling and suggest clinical implications of circulating TGF-ß-high EVs as a potential biomarker of HIV-associated PH.

The Potential Role of Extracellular Vesicles in COVID-19 Associated Endothelial injury and Pro-inflammation.

COVID-19 infection caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic with the number of deaths growing exponentially. Early evidence points to significant endothelial dysfunction, micro-thromboses, pro-inflammation as well as a dysregulated immune response in the pathogenesis of this disease. In this study, we analyzed the cargo of EVs isolated from the plasma of patients with COVID-19 for the identification of potential biomarkers of disease severity and to explore their role in disease pathogenesis. Plasma-derived EVs were isolated from 53 hospitalized patients with COVID infection and compared according to the severity of the disease. Analysis of inflammatory and cardiovascular protein cargo of large EVs revealed significantly differentially expressed proteins for each disease sub-group. Notably, members of the TNF superfamily and IL-6 family were up-regulated in patients on oxygen support with severe and moderate disease. EVs from the severe group were also enhanced with pro-thrombotic/endothelial injury factors (TF, t-PA, vWF) and proteins associated with cardiovascular pathology (MB, PRSS8, REN, HGF). Significantly higher levels of TF, CD163, and EN-RAGE were observed in EVs from severe patients when compared to patients with a moderate disease requiring supplemental O2. Importantly, we also observed increased caspase 3/7 activity and decreased cell survival in human pulmonary microvascular endothelial cells exposed to EVs from the plasma of patients with severe disease compared to healthy controls. In conclusion, our findings indicate alterations in pro-inflammatory, coagulopathy, and endothelial injury protein cargo in large EVs in response to SARS-CoV-2 infection that may be a causative agent in severe illness.

Extracellular vesicles: novel communicators in lung diseases.

The lung is the organ with the highest vascular density in the human body. It is therefore perceivable that the endothelium of the lung contributes significantly to the circulation of extracellular vesicles (EVs), which include exosomes, microvesicles, and apoptotic bodies. In addition to the endothelium, EVs may arise from alveolar macrophages, fibroblasts and epithelial cells. Because EVs harbor cargo molecules, such as miRNA, mRNA, and proteins, these intercellular communicators provide important insight into the health and disease condition of donor cells and may serve as useful biomarkers of lung disease processes. This comprehensive review focuses on what is currently known about the role of EVs as markers and mediators of lung pathologies including COPD, pulmonary hypertension, asthma, lung cancer and ALI/ARDS. We also explore the role EVs can potentially serve as therapeutics for these lung diseases when released from healthy progenitor cells, such as mesenchymal stem cells.

NADPH oxidase-mediated endothelial injury in HIV- and opioid-induced pulmonary arterial hypertension.

We previously demonstrated that the combined exposure of human pulmonary microvascular endothelial cells (HPMECs) to morphine and viral protein(s) results in the oxidative stress-mediated induction of autophagy, leading to shift in the cells from early apoptotic to apoptosis-resistant proliferative status associated with the angioproliferative remodeling observed in pulmonary arterial hypertension (PAH). In this study, we tried to delineate the major source of HIV-1 protein Tat and morphine induced oxidative burst in HPMECs and its consequences on vascular remodeling and PAH in an in vivo model. We observed switch from the initial increased expression of NADPH oxidase (NOX) 2 in response to acute treatment of morphine and HIV-Tat to later increased expression of NOX4 on chronic treatment in the endoplasmic reticulum of HPMECs without any alterations in the mitochondria. Furthermore, NOX-dependent induction of autophagy was observed to play a pivotal role in regulating the endothelial cell survival. Our in vivo findings showed significant increase in pulmonary vascular remodeling, right ventricular systolic pressure, and Fulton index in HIV-transgenic rats on chronic administration of morphine. This was associated with increased oxidative stress in lung tissues and rat pulmonary microvascular endothelial cells. Additionally, endothelial cells from morphine-treated HIV-transgenic rats demonstrated increased expression of NOX2 and NOX4 proteins, inhibition of which ameliorated their increased survival upon serum starvation. In conclusion, this study describes NADPH oxidases as one of the main players in the oxidative stress-mediated endothelial dysfunction on the dual hit of HIV-viral protein(s) and opioids.

Sugen-morphine model of pulmonary arterial hypertension.

Pulmonary arterial hypertension is a fatal disease associated with pulmonary vascular remodeling and right ventricular hypertrophy. Pre-clinical animal models that reproduce the human pulmonary arterial hypertension process and pharmacological response to available therapies are critical for future drug development. The most prevalent animal model reproducing many aspects of angioobliterative forms of pulmonary arterial hypertension is the rat Sugen/hypoxia model in which Sugen, a vascular endothelial growth factor receptor antagonist, primarily causes initiation of endothelial injury and later in the presence of hypoxia promotes proliferation of apoptosis-resistant endothelial cells. We previously demonstrated that exposure of human pulmonary microvascular endothelium to morphine and HIV-proteins results in initial apoptosis followed by increased proliferation. Here, we demonstrate that the double-hit of morphine and Sugen 5416 (Sugen-morphine) in rats leads to the development of pulmonary arterial hypertension with significant medial hypertrophy of pre-acinar pulmonary arteries along with neo-intimal thickening of intra-acinar vessels. In addition, the pulmonary smooth muscle and endothelial cells isolated from Sugen-morphine rats showed hyperproliferation and apoptotic resistance, respectively, in response to serum starvation. Our findings support that the dual hit model of Sugen 5416 and morphine provides another experimental strategy to induce significant pulmonary vascular remodeling and development of severe pulmonary arterial hypertension pathology in rats without exposure to hypoxia.

Analysis of lncRNA-miRNA-mRNA Interactions in Hyper-proliferative Human Pulmonary Arterial Smooth Muscle Cells.

We previously reported enhanced proliferation of smooth muscle cells on the combined exposure of HIV proteins and cocaine leading to the development of HIV-pulmonary arterial hypertension. Here, we attempt to comprehensively understand the interactions between long noncoding RNAs (lncRNAs), mRNAs and micro-RNAs (miRNAs) to determine their role in smooth muscle hyperplasia. Differential expression of lncRNAs, mRNAs and miRNAs were obtained by microarray and small-RNA sequencing from HPASMCs treated with and without cocaine and/or HIV-Tat. LncRNA to mRNA associations were conjectured by analyzing their genomic proximity and by interrogating their association to vascular diseases and cancer co-expression patterns reported in the relevant databases. Neuro-active ligand receptor signaling, Ras signaling and PI3-Akt pathway were among the top pathways enriched in either differentially expressed mRNAs or mRNAs associated to lncRNAs. HPASMC with combined exposure to cocaine and Tat (C + T) vs control identified the following top lncRNA-mRNA pairs, ENST00000495536-HOXB13, T216482-CBL, ENST00000602736-GDF7, and, TCONS_00020413-RND1. Many of the down-regulated miRNAs in the HPASMCs treated with C + T were found to be anti-proliferative and targets of up-regulated lncRNAs targeting up-regulated mRNAs, including down-regulation of miR-185, -491 and up-regulation of corresponding ENST00000585387. Specific knock down of the selected lncRNAs highlighted the importance of non-coding RNAs in smooth muscle hyperplasia.

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death.

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.

Drug abuse and HIV-related pulmonary hypertension: double hit injury.

: Improved survival among HIV-1-infected individuals with the advent of antiretroviral therapy has clearly led to a greater prevalence of noninfectious complications. One of the most devastating sequelae in these individuals is the development of pulmonary arterial hypertension (PAH). Various epidemiological studies suggest worse survival of HIV-PAH patients when compared with other forms of PAH. Given that only a subset and not all HIV-infected individuals develop HIV-PAH, it is suggested that an additional second-hit of genetic or environmental trigger is needed for the development of PAH. In this context, it has been well documented that HIV patients who abuse illicit drugs such as stimulants, opioids, and the like, are more susceptible to develop PAH. In this review, we highlight the studies that support the significance of a double hit of HIV and drug abuse in the incidence of PAH and focus on the research that has been undertaken to unravel the pathobiology and vascular remodeling mechanisms underlying the deleterious synergy between HIV infection and drugs of abuse in orchestrating the development of PAH.

Macrophage-derived extracellular vesicles mediate smooth muscle hyperplasia: role of altered miRNA cargo in response to HIV infection and substance abuse.

Our previous studies consistently demonstrate enhanced pulmonary vascular remodeling in HIV-infected intravenous drug users, and in simian immunodeficiency virus-infected macaques or HIV-transgenic rats exposed to opioids or cocaine. Although we reported an associated increase in perivascular inflammation, the exact role of inflammatory cells in the development of pulmonary vascular remodeling remains unknown. In this study, HIV-infected and cocaine (H+C)-treated human monocyte derived macrophages released a higher number of extracellular vesicles (EVs), compared to HIV-infected or uninfected cocaine-treated macrophages, with a significant increase in the particle size range to 100-150 nm. Treatment of primary human pulmonary arterial smooth muscle cells (HPASMCs) with these EVs resulted in a significant increase in smooth muscle proliferation. We also observed a significant increase in the miRNA-130a level in the EVs derived from H+C-treated macrophages that corresponded with the decrease in the expression of phosphatase and tensin homolog and tuberous sclerosis 1 and 2 and activation of PI3K/protein kinase B signaling in HPASMCs on addition of these EVs. Transfection of HPASMCs with antagomir-130a-ameliorated the EV-induced effect. Thus, we conclude that EVs derived from H+C-treated macrophages promote pulmonary smooth muscle proliferation by delivery of its prosurvival miRNA cargo, which may play a crucial role in the development of PAH.-Sharma, H., Chinnappan, M., Agarwal, S., Dalvi, P., Gunewardena, S., O'Brien-Ladner, A., Dhillon, N. K. Macrophage-derived extracellular vesicles mediate smooth muscle hyperplasia: role of altered miRNA cargo in response to HIV infection and substance abuse.

Network of MicroRNAs Mediate Translational Repression of Bone Morphogenetic Protein Receptor-2: Involvement in HIV-Associated Pulmonary Vascular Remodeling.

BACKGROUND: Earlier, we reported that the simultaneous exposure of pulmonary arterial smooth muscle cells to HIV proteins and cocaine results in the attenuation of antiproliferative bone morphogenetic protein receptor-2 (BMPR2) protein expression without any decrease in its mRNA levels. Therefore, in this study, we aimed to investigate the micro RNA-mediated posttranscriptional regulation of BMPR2 expression. METHODS AND RESULTS: We identified a network of BMPR2 targeting micro RNAs including miR-216a to be upregulated in response to cocaine and Tat-mediated augmentation of oxidative stress and transforming growth factor-ß signaling in human pulmonary arterial smooth muscle cells. By using a loss or gain of function studies, we observed that these upregulated micro RNAs are involved in the Tat- and cocaine-mediated smooth muscle hyperplasia via regulation of BMPR2 protein expression. These in vitro findings were further corroborated using rat pulmonary arterial smooth muscle cells isolated from HIV transgenic rats exposed to cocaine. More importantly, luciferase reporter and in vitro translation assays demonstrated that direct binding of novel miR-216a and miR-301a to 3'UTR of BMPR2 results in the translational repression of BMPR2 without any degradation of its mRNA. CONCLUSIONS: We identified for the first time miR-216a as a negative modulator of BMPR2 translation and observed it to be involved in HIV protein(s) and cocaine-mediated enhanced proliferation of pulmonary smooth muscle cells.

Drug-induced pulmonary arterial hypertension: a primer for clinicians and scientists.

Drug-induced pulmonary arterial hypertension (D-PAH) is a form of World Health Organization Group 1 pulmonary hypertension (PH) defined by severe small vessel loss and obstructive vasculopathy, which leads to progressive right heart failure and death. To date, 16 different compounds have been associated with D-PAH, including anorexigens, recreational stimulants, and more recently, several Food and Drug Administration-approved medications. Although the clinical manifestation, pathology, and hemodynamic profile of D-PAH are indistinguishable from other forms of pulmonary arterial hypertension, its clinical course can be unpredictable and to some degree dependent on removal of the offending agent. Because only a subset of individuals develop D-PAH, it is probable that genetic susceptibilities play a role in the pathogenesis, but the characterization of the genetic factors responsible for these susceptibilities remains rudimentary. Besides aggressive treatment with PH-specific therapies, the major challenge in the management of D-PAH remains the early identification of compounds capable of injuring the pulmonary circulation in susceptible individuals. The implementation of pharmacovigilance, precision medicine strategies, and global warning systems will help facilitate the identification of high-risk drugs and incentivize regulatory strategies to prevent further outbreaks of D-PAH. The goal for this review is to inform clinicians and scientists of the prevalence of D-PAH and to highlight the growing number of common drugs that have been associated with the disease.

Hyperactive TGF-ß Signaling in Smooth Muscle Cells Exposed to HIV-protein(s) and Cocaine: Role in Pulmonary Vasculopathy.

We earlier demonstrated synergistic increase in the proliferation of pulmonary smooth muscle cells on exposure to HIV-proteins and/or cocaine due to severe down-modulation of bone morphogenetic protein receptor (BMPR) axis: the anti-proliferative arm of TGF-ß super family of receptors. Here, now we demonstrate the effect of HIV-Tat and cocaine on the proliferative TGF-ß signaling cascade. We observed a significant increase in the secretion of TGF-ß1 ligand along with enhanced protein expression of TGFß Receptor (TGFßR)-1, TGFßR-2 and phosphorylated SMAD2/3 in human pulmonary arterial smooth muscle cells on treatment with cocaine and Tat. Further, we noticed an increase in the levels of p-TAK1 complexed with TGFßR-2. Concomitant to this a significant increase in the activation of TAK1-mediated, SMAD-independent downstream signaling molecules: p-MKK4 and p-JNK was observed. However, activation of MKK3/6-p38MAPK, another axis downstream of TAK1 was found to be reduced due to attenuation in the protein levels of BMPR2. Both SMAD and non-SMAD dependent TGFßR cascades were found to contribute to hyper-proliferation. Finally the increase in the levels of phosphorylated TGFßR1 and TGFßR2 on exposure to HIV-proteins and cocaine was confirmed in pulmonary smooth muscle cells from cocaine injected HIV-transgenic rats and in total lung extracts from HIV infected cocaine and/or opioid users.