Pranjali Dalvi

Enhanced Pulmonary Arteriopathy in Simian Immunodeficiency Virus-infected Macaques Exposed to Morphine

RATIONALE HIV-associated pulmonary arterial hypertension (PAH) is likely a more prevalent noninfectious complication of AIDS than previously recognized. Furthermore, the majority of HIV-PAH cases occur in individuals with a history of intravenous drug use. In this study we used a simian immunodeficiency (SIV) macaque model and a primary cell-culture system to investigate the association between drug abuse and HIV infection in HIV-PAH development. METHODS The archival lung tissues from macaques previously used to study the effect of morphine on SIV infection-associated neuropathogenesis were analyzed for pulmonary vascular changes. The direct effect of HIV proteins and illicit drugs was investigated on oxidative stress, survival, and proliferation of human pulmonary microvascular endothelial cells. MEASUREMENTS AND MAIN RESULTS SIVmacR71/17E-infected rhesus macaques treated with morphine (VM group) demonstrated significant pulmonary vascular remodeling, including the presence of early and advanced complex (plexiform) lesions, when compared with either the SIV-infected (V group) or morphine-treated uninfected (M group) macaques. However, both the V (two of five) and VM (two of six) groups included some animals with Pneumocystis jirovecii pneumonia. The endothelial cells lining the vessels with medial hypertrophy or initial-stage intimal lesions in lung sections from VM macaques demonstrated an increase in positivity for both terminal dUTP nick-end labeling and Ki67. Oxidative stress-mediated enhanced apoptosis followed by enhanced proliferation of endothelial cells was observed on simultaneous treatment with viral proteins and drugs of abuse compared with either treatment alone. CONCLUSIONS Our findings suggest that SIV/HIV protein(s) and morphine interact to cause the proliferation of apoptosis-resistant endothelial cells leading to angio-obliteration.

HIV-1/Cocaine Induced Oxidative Stress Disrupts Tight Junction Protein-1 in Human Pulmonary Microvascular Endothelial Cells: Role of Ras/ERK1/2 Pathway

Intravenous drug use (IVDU) is the major risk factor in the development of HIV-related pulmonary arterial hypertension (HRPAH); however, the pathogenesis of HRPAH in association with IVDU has yet to be characterized. Endothelial injury is considered to be an initiating factor for pulmonary vascular remodeling in animal models of PAH. Our previous study shows that simultaneous exposure to HIV-Trans-activator of transcription (Tat) and cocaine exacerbates both disruption of tight junction proteins and permeability of human pulmonary artery endothelial cells compared with either treatment alone. We here now demonstrate that this HIV-Tat and cocaine mediated endothelial dysfunction accompanies with increase in hydrogen peroxide and superoxide radicals generation and involves redox sensitive signaling pathway. Pretreatment with antioxidant cocktail attenuated the cocaine and Tat mediated disassembly of Zonula Occludens (ZO)-1 and enhancement of endothelial monolayer permeability. Furthermore, inhibition of NADPH oxidase by apocynin or siRNA-mediated knockdown of gp-91phox abolished the Tat/cocaine-induced reactive oxygen species (ROS) production, suggesting the NADPH oxidase mediated generation of oxidative radicals. In addition, ROS dependent activation of Ras and ERK1/2 Kinase was observed to be mediating the TJP-1 disassembly, and endothelial dysfunction in response to cocaine and Tat exposure. In conclusion, our findings demonstrate that Tat/cocaine -mediated production of ROS activate Ras/Raf/ERK1/2 pathway that contributes to disruption of tight junction protein leading to pulmonary endothelial dysfunction associated with pulmonary vascular remodeling.

Downregulation of Bone Morphogenetic Protein Receptor Axis During HIV-1 and Cocaine-Mediated Pulmonary Smooth Muscle Hyperplasia Implications for HIV-Related Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is one of the most common non-infectious complications of HIV-infection1 with approximately 1000 times higher incidence in HIV-infected patients compared with the general population2. The probability of survival reduces to one half in the individuals who develop HIV-related PAH (HRPAH) compared with HIV-infected individuals without PAH3. Despite major clinical advances in therapy over the past few years, the prognosis of HRPAH remains poor and is similar to that of some advanced cancers. Furthermore, while it is evident from other case reports that the abuse of cocaine and other stimulants is a possible risk factor in the development of PAH4-6, intravenous drug use (IVDU) was found to be one of the major risk factors for HIV-infection in the HRPAH patients7. Our recent study showing enhanced pulmonary vascular remodeling in HIV-infected lung tissues from IV heroin and/or cocaine abusers indicates that IVDU and HIV-1 potentially act in concert to cause pulmonary arteriopathy8. However, it is still not clear how illicit drugs and HIV-infection either alone or in combination can cause the vascular dys-regulation associated with increased pulmonary vascular resistance and cardiac dysfunction. The possibility of direct HIV-infection of pulmonary vasculature cells leading to HRPAH development is unlikely since HIV-1 RNA or DNA is not found in the pulmonary vessels of human lung tissues9. Studies demonstrate that the direct action of HIV-proteins released by the infected lymphocytes and macrophages play a major role in the development of HRPAH10. Recently, we11 showed that pulmonary vascular remodeling develops in the presence of HIV-1 proteins without an active infection, leading to pulmonary hypertension1 in a non-infectious HIV-transgenic rat model. The pulmonary arterial smooth muscle cells (PASMCs) are key players in the pathogenesis of all forms of PAH vascular remodeling. The exposure of PASMCs to viral proteins and growth factors after damage to the endothelial monolayer leads to smooth muscle hypertrophy and proliferation. However, the cellular and molecular mechanisms underlying the thickening of blood vessels are poorly defined. While bone morphogenetic protein receptor (BMPR)-2 mutations have been associated with familial PAH; many studies suggest that a critical reduction in the expression of BMPRs may be important in the pathogenesis of PAH12. Bone morphogenetic protein-2 or -4 on binding to BMPR negatively regulates smooth muscle cell growth and proliferation13. The BMP ligands bind to heteromeric complexes of BMPR-1A or BMPR-1B with BMPR-2 resulting in the phosphorylation of Regulatory-Sma and MAD Related Family proteins (R-SMAD). Activated RSMADs: SMAD-1/5/8 then form complex with SMAD4 that translocate to the nucleus and regulate the transcription of BMP/SMAD-responsive anti-proliferative genes14. HIV-protein Tat, the transactivating factor of HIV-1 is actively secreted by infected cells15 and acts as an angiogenic and oncogenic factor by promoting growth, migration and production of growth factors in various cell-types12, 15. In our previous findings, we have shown that cocaine synergizes with HIV-Tat to promote proliferation of PASMCs8. In this study we partially defined the mechanism(s) mediating this increased proliferation and enhanced pulmonary vascular remodeling in HIV-infected IVDUs by examining the alterations in the anti-proliferative BMP/BMPR axis. We here report significantly more attenuation in the BMPR protein expression in PASMCs on combined treatment with HIV-Tat and cocaine compared to either treatment alone, concomitant with abrogation of BMPR downstream signaling and anti- proliferative Id1 gene expression. Furthermore, to the best of our knowledge this is the first report demonstrating significant down-modulation of BMPR expression in the lungs from HIV infected IVDUs compared to HIV-infected non-drug users or un-infected IVDUs. Some of the results of these studies have been previously reported in the form of abstract.Objective—Our previous findings support an additive effect of cocaine to HIV infection in the development of pulmonary arteriopathy through enhanced proliferation of human pulmonary smooth muscle cells. We now examined the role of antiproliferative bone morphogenetic protein receptor (BMPR) axis in HIV protein and cocaine-mediated pulmonary smooth muscle hyperplasia. Approach and Results—Stimulation of BMPR axis resulted in attenuation of synergistic increase in the proliferation of human pulmonary arterial smooth muscle cells in response to cocaine and HIV protein, transactivator of transcription (Tat). Interestingly, an increase in mRNA but decrease in protein levels of BMPR with correlated decrease in the activation of Sma- and MAD-related family protein 1/5/8 and Id1 gene expression was observed on combined treatment with cocaine and Tat when compared with the untreated cells at all time points tested. Although longer exposure to either cocaine or Tat alone also resulted in a significant decrease in the BMPR protein expression, the abrogation on combined treatment was still significantly more when compared with that of the monotreatments. Significant increase in mRNA but downmodulation of BMPR protein expression was also observed in the lung extracts from HIV-infected intravenous drug users (HIV+IVDU) when compared with that from HIV-infected non-IVDUs (HIV) or uninfected IVDUs (IVDU). Furthermore, significant decrease in BMPR protein expression was also observed in HIV or IVDUs when compared with normal controls that correlated with in vitro findings on chronic exposure to cocaine or HIV protein alone. Conclusions—Simultaneous exposure of pulmonary smooth muscle cells to viral protein(s) and cocaine exacerbates downregulation of BMPR axis that may result in enhanced pulmonary vasculature aberrations in HIV+IVDUs.

Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells.

The primary objective of this study is to functionally characterize and provide molecular evidence of large neutral amino acid transporter (LAT1) in human derived prostate cancer cells (PC-3). We carried out the uptake of [3H]-tyrosine to assess the functional activity of LAT1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis is carried out to confirm the molecular expression of LAT1. [3H]-tyrosine uptake is found to be time dependent and linear up to 60 min. The uptake process does not exhibit any dependence on sodium ions, pH and energy. However, it is temperature dependent and found maximal at physiological temperature. The uptake of [3H]-tyrosine demonstrates saturable kinetics with K(m) and V(max) values of 34 ± 3 μM and 0.70 ± 0.02 nanomoles/min/mg protein, respectively. It is strongly inhibited by large neutral (phenylalanine, tryptophan, leucine, isoleucine) and small neutral (alanine, serine, cysteine) but not by basic (lysine and arginine) and acidic (aspartic and glutamic acid) amino acids. Isoleucine-quinidine (Ile-quinidine) prodrug generates a significant inhibitory effect on [3H]-tyrosine uptake suggesting that it is recognized by LAT1. RT-PCR analysis provided a product band at 658 and 840 bp, specific to LAT1 and LAT2, respectively. For the first time, this study demonstrates that LAT1, primarily responsible for the uptake of large neutral amino acids, is functionally active in PC-3 cells. Significant increase in the uptake generated by Ile-quinidine relative to quinidine suggests that LAT1 can be utilized for enhancing the cellular permeation of poor cell permeable anticancer drugs. Furthermore, this cell line can be utilized as an excellent in vitro model for studying the interaction of large neutral amino acid conjugated drugs with LAT1 transporter.

Down-regulation of BMPR Axis during HIV-1 and Cocaine-mediated Pulmonary Smooth Muscle Hyperplasia: Implications for HRPAH

Pulmonary arterial hypertension is one of the most common non-infectious complications of HIV-infection1 with approximately 1000 times higher incidence in HIV-infected patients compared with the general population2. The probability of survival reduces to one half in the individuals who develop HIV-related PAH (HRPAH) compared with HIV-infected individuals without PAH3. Despite major clinical advances in therapy over the past few years, the prognosis of HRPAH remains poor and is similar to that of some advanced cancers. Furthermore, while it is evident from other case reports that the abuse of cocaine and other stimulants is a possible risk factor in the development of PAH4-6, intravenous drug use (IVDU) was found to be one of the major risk factors for HIV-infection in the HRPAH patients7. Our recent study showing enhanced pulmonary vascular remodeling in HIV-infected lung tissues from IV heroin and/or cocaine abusers indicates that IVDU and HIV-1 potentially act in concert to cause pulmonary arteriopathy8. However, it is still not clear how illicit drugs and HIV-infection either alone or in combination can cause the vascular dys-regulation associated with increased pulmonary vascular resistance and cardiac dysfunction. The possibility of direct HIV-infection of pulmonary vasculature cells leading to HRPAH development is unlikely since HIV-1 RNA or DNA is not found in the pulmonary vessels of human lung tissues9. Studies demonstrate that the direct action of HIV-proteins released by the infected lymphocytes and macrophages play a major role in the development of HRPAH10. Recently, we11 showed that pulmonary vascular remodeling develops in the presence of HIV-1 proteins without an active infection, leading to pulmonary hypertension1 in a non-infectious HIV-transgenic rat model. The pulmonary arterial smooth muscle cells (PASMCs) are key players in the pathogenesis of all forms of PAH vascular remodeling. The exposure of PASMCs to viral proteins and growth factors after damage to the endothelial monolayer leads to smooth muscle hypertrophy and proliferation. However, the cellular and molecular mechanisms underlying the thickening of blood vessels are poorly defined. While bone morphogenetic protein receptor (BMPR)-2 mutations have been associated with familial PAH; many studies suggest that a critical reduction in the expression of BMPRs may be important in the pathogenesis of PAH12. Bone morphogenetic protein-2 or -4 on binding to BMPR negatively regulates smooth muscle cell growth and proliferation13. The BMP ligands bind to heteromeric complexes of BMPR-1A or BMPR-1B with BMPR-2 resulting in the phosphorylation of Regulatory-Sma and MAD Related Family proteins (R-SMAD). Activated RSMADs: SMAD-1/5/8 then form complex with SMAD4 that translocate to the nucleus and regulate the transcription of BMP/SMAD-responsive anti-proliferative genes14. HIV-protein Tat, the transactivating factor of HIV-1 is actively secreted by infected cells15 and acts as an angiogenic and oncogenic factor by promoting growth, migration and production of growth factors in various cell-types12, 15. In our previous findings, we have shown that cocaine synergizes with HIV-Tat to promote proliferation of PASMCs8. In this study we partially defined the mechanism(s) mediating this increased proliferation and enhanced pulmonary vascular remodeling in HIV-infected IVDUs by examining the alterations in the anti-proliferative BMP/BMPR axis. We here report significantly more attenuation in the BMPR protein expression in PASMCs on combined treatment with HIV-Tat and cocaine compared to either treatment alone, concomitant with abrogation of BMPR downstream signaling and anti- proliferative Id1 gene expression. Furthermore, to the best of our knowledge this is the first report demonstrating significant down-modulation of BMPR expression in the lungs from HIV infected IVDUs compared to HIV-infected non-drug users or un-infected IVDUs. Some of the results of these studies have been previously reported in the form of abstract.Objective—Our previous findings support an additive effect of cocaine to HIV infection in the development of pulmonary arteriopathy through enhanced proliferation of human pulmonary smooth muscle cells. We now examined the role of antiproliferative bone morphogenetic protein receptor (BMPR) axis in HIV protein and cocaine-mediated pulmonary smooth muscle hyperplasia. Approach and Results—Stimulation of BMPR axis resulted in attenuation of synergistic increase in the proliferation of human pulmonary arterial smooth muscle cells in response to cocaine and HIV protein, transactivator of transcription (Tat). Interestingly, an increase in mRNA but decrease in protein levels of BMPR with correlated decrease in the activation of Sma- and MAD-related family protein 1/5/8 and Id1 gene expression was observed on combined treatment with cocaine and Tat when compared with the untreated cells at all time points tested. Although longer exposure to either cocaine or Tat alone also resulted in a significant decrease in the BMPR protein expression, the abrogation on combined treatment was still significantly more when compared with that of the monotreatments. Significant increase in mRNA but downmodulation of BMPR protein expression was also observed in the lung extracts from HIV-infected intravenous drug users (HIV+IVDU) when compared with that from HIV-infected non-IVDUs (HIV) or uninfected IVDUs (IVDU). Furthermore, significant decrease in BMPR protein expression was also observed in HIV or IVDUs when compared with normal controls that correlated with in vitro findings on chronic exposure to cocaine or HIV protein alone. Conclusions—Simultaneous exposure of pulmonary smooth muscle cells to viral protein(s) and cocaine exacerbates downregulation of BMPR axis that may result in enhanced pulmonary vasculature aberrations in HIV+IVDUs.

Effect of Cocaine on Pulmonary Vascular Remodeling and Hemodynamics in Human Immunodeficiency Virus–Transgenic Rats

Human immunodeficiency virus (HIV)-related pulmonary arterial hypertension has been found to be more prevalent in intravenous drug users. Our earlier cell-culture findings reported down-regulation of bone morphogenetic protein receptors (BMPRs) in combination with enhanced proliferation of human pulmonary arterial smooth muscle cells (PASMCs) in the presence of HIV-Trans-activator of transcription (Tat) and cocaine compared with either treatment alone. Here, we report physiologic evidence of significant increases in mean pulmonary arterial pressure in HIV-transgenic (Tg) rats intraperitoneally administered 40 mg/kg body weight cocaine (HIV-cocaine group) once daily for 21 days when compared with HIV-Tg rats given saline (HIV group) or wild-type (WT) Fischer 334 rats treated with (WT-cocaine group) and without cocaine (WT group). In addition, right ventricle systolic pressure was also found to be significantly higher in the HIV-cocaine rats compared with the WT group. Significant down-regulation in protein expression of BMPR-2 and BMPR-1B was observed in total lung extract from HIV-cocaine rats compared with the other three groups. Furthermore, the PASMCs isolated from HIV-cocaine rats demonstrated a higher level of proliferation and lower levels of apoptosis compared with cells isolated from other rat groups. Interestingly, corroborating our earlier cell-culture findings, we observed higher expression of BMPR-2 and BMPR-1B messenger RNA and significantly lower levels of BMPR-2 and BMPR-1B protein in HIV-cocaine PASMCs compared with cells isolated from all other groups. In conclusion, our findings support an additive effect of cocaine and HIV on smooth muscle dysfunction, resulting in enhanced pulmonary vascular remodeling with associated elevation of mean pulmonary arterial pressure and right ventricle systolic pressure in HIV-Tg rats exposed to cocaine.

Enhanced autophagy in pulmonary endothelial cells on exposure to HIV-Tat and morphine: Role in HIV-related pulmonary arterial hypertension

ABSTRACT Intravenous drug use is one of the major risk factors for HIV-infection in HIV-related pulmonary arterial hypertension patients. We previously demonstrated exaggerated pulmonary vascular remodeling with enhanced apoptosis followed by increased proliferation of pulmonary endothelial cells on simultaneous exposure to both opioids and HIV protein(s). Here we hypothesize that the exacerbation of autophagy may be involved in the switching of endothelial cells from an early apoptotic state to later hyper-proliferative state. Treatment of human pulmonary microvascular endothelial cells (HPMECs) with both the HIV-protein Tat and morphine resulted in an oxidative stress-dependent increase in the expression of various markers of autophagy and formation of autophagosomes when compared to either Tat or morphine monotreatments as demonstrated by western blot, transmission electron microscopy and immunofluorescence. Autophagy flux experiments suggested increased formation rather than decreased clearance of autolysosomes. Inhibition of autophagy resulted in a significant increase in apoptosis and reduction in proliferation of HPMECs with combined morphine and Tat (M+T) treatment compared to monotreatments whereas stimulation of autophagy resulted in opposite effects. Significant increases in the expression of autophagy markers as well as the number of autophagosomes and autolysosomes was observed in the lungs of SIV-infected macaques and HIV-infected humans exposed to opioids. Overall our findings indicate that morphine in combination with viral protein(s) results in the induction of autophagy in pulmonary endothelial cells that may lead to an increase in severity of angio-proliferative remodeling of the pulmonary vasculature on simian and human immunodeficiency virus infection in the presence of opioids.

Ligand-Independent Activation of Platelet-Derived Growth Factor Receptor β during Human Immunodeficiency Virus–Transactivator of Transcription and Cocaine-Mediated Smooth Muscle Hyperplasia

Our previous study supports an additive effect of cocaine to human immunodeficiency virus infection in the development of pulmonary arteriopathy through enhancement of proliferation of pulmonary smooth muscle cells (SMCs), while also suggesting involvement of platelet-derived growth factor receptor (PDGFR) activation in the absence of further increase in PDGF-BB ligand. Redox-related signaling pathways have been shown to regulate tyrosine kinase receptors independent of ligand binding, so we hypothesized that simultaneous treatment of SMCs with transactivator of transcription (Tat) and cocaine may be able to indirectly activate PDGFR through modulation of reactive oxygen species (ROS) without the need for PDGF binding. We found that blocking the binding of ligand using suramin or monoclonal IMC-3G3 antibody significantly reduced ligand-induced autophosphorylation of Y1009 without affecting ligand-independent transphosphorylation of Y934 residue on PDGFRβ in human pulmonary arterial SMCs treated with both cocaine and Tat. Combined treatment of human pulmonary arterial SMCs with cocaine and Tat resulted in augmented production of superoxide radicals and hydrogen peroxide when compared with either treatment alone. Inhibition of this ROS generation prevented cocaine- and Tat-mediated Src activation and transphosphorylation of PDGFRβ at Y934 without any changes in phosphorylation of Y1009, in addition to attenuation of smooth muscle hyperplasia. Furthermore, pretreatment with an Src inhibitor, PP2, also suppressed cocaine- and Tat-mediated enhanced Y934 phosphorylation and smooth muscle proliferation. Finally, we report total abrogation of cocaine- and Tat-mediated synergistic increase in cell proliferation on inhibition of both ligand-dependent and ROS/Src-mediated ligand-independent phosphorylation of PDGFRβ.

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‐β 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.