Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling
© Mermis et al; licensee BioMed Central Ltd. 2011
Received: 23 May 2011
Accepted: 5 August 2011
Published: 1 December 2011
Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.
The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.
HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.
In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.
The advent of antiretroviral therapy (ART) has clearly led to improved survival among HIV-1 infected individuals, yet this advancement has resulted in the unexpected consequence of virus-associated noninfectious complications such as HIV-related pulmonary arterial hypertension (HIV-PAH) [1, 2]. Despite adherence with ART, development of HIV-PAH serves as an independent predictor of death in patients with HIV-infection . A precise characterization of the pathogenesis of HIV-PAH has so far proven elusive. As there is little evidence for direct viral infection within the pulmonary vascular bed [4–7], popular hypothesis is that secretary HIV-1 viral proteins in circulation are capable of inducing vascular oxidative stress and direct endothelial cell dysfunction and smooth muscle cell proliferation critical to the development of HIV-related arteriopathy [8, 9]. Further, evidence is accumulating which suggests that the HIV-1 infection of monocyte/macrophages and lymphocytes stimulates increased production of pro-inflammatory markers and/or growth factors. implicated in the pathogenesis of HIV-PAH such as platelet derived growth factor (PDGF)-BB [10–16]. These soluble mediators can then initiate endothelial injury followed by smooth muscle cell proliferation and migration [2, 17, 18].
Previous studies provide evidence for the possible involvement of PDGF in the pathogenesis of pulmonary vascular remodeling in animal models [19, 20] and in lung biopsies from patients with PPH or with HIV-PAH . Furthermore, a non-specific inhibitor of PDGF signaling, imatinib, has demonstrated the ability to diminish vascular remodeling in animal studies and to mitigate clinical decline in human PAH trials [21–24]. Our previous work demonstrates an over-expression of PDGF in-vitro in HIV-infected macrophages  and in-vivo in Simian HIV-infected macaques . Our recent work supports an HIV-protein mediated up-regulation of PDGF-BB in un-infectable vascular cell types such as human primary pulmonary arterial endothelial and smooth muscle cells . However, the mechanism(s) by which HIV infection or viral protein(s) binding induces PDGF expression and the role of this potent mitogen in the setting of HIV-associated pulmonary arteriopathy has not been well characterized. HIV associated viral proteins including Tat and gp-120 have demonstrated the ability to trigger the generation of reactive oxygen species (ROS) [27, 28]. As oxidative stress stabilizes hypoxia inducible factor (HIF)-1α, a transcription factor critical for regulation of important proliferative and vaso-active mediators [29–31], we hypothesize that viral protein generated reactive oxygen species (ROS) induce HIF-1α accumulation, with a resultant enhanced transcription of PDGF-B chain.
Thus, given the need for clarification of the mechanisms responsible for HIV-related pulmonary vascular remodeling, we, in the present study, first utilized the non-infectious NL4-3Δgag/pol HIV-1 transgenic (HIV-Tg) rat model [32, 33] to explore the direct role of viral proteins in the development of pulmonary vascular remodeling. This HIV-Tg rat model , develops many clinical multisystem manifestations similar to those found in AIDS patients and most importantly, has earlier been demonstrated to be under significant oxidative stress. Furthermore, given that the pulmonary artery endothelial dysfunction plays a key role in the initiation and progression of PAH [35–37], utilizing the primary pulmonary endothelial cell-culture system we next delineated the importance of oxidative stress and HIF-1α activation in viral protein mediated up-regulation of PDGF-BB.
HIV-1 transgenic and wild type rats
HIV-1 transgenic (Tg) Sprague Dawley (SD) and SD wild type (WT) rats were purchased from Harlan (Indianapolis, Indiana). Young 4-5 months old Tg rats (n = 6) and age matched SD wild type rats (n = 6) were used for analysis. The HIV-1 Tg rat contains a gag-pol deleted NL4-3 provirus and expresses HIV viral RNA and proteins in various tissues including lymphocytes and monocytes. The animals were euthanized with inhalation of 2.5-3% isofluorane gas, followed by transcardial saline perfusion. Following euthanasia, one half of the lung was post-fixed for histological examination, while the other half was snap frozen for RNA analysis. The animal care at the Kansas University Medical Center was in strict accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Right Ventricular Mass Evaluation
Hearts were removed from the euthanized animals. After the removal of atria, the wall of the right ventricle (RV) was separated from the left ventricle (LV) and septum (LV+S) according to the established method . Wet weights of both RV & LV+S were quantified, normalized to the total body weight and used to calculate the RV/LV+S ratio.
Histology and immuno-histochemical analysis of pulmonary arteries
Excised lungs were immersed in 4% paraformaldehyde overnight followed by 70% ethanol and then used for paraffin embedding. Paraffin sections of 5 μm thickness were used for Hematoxylin & Eosin (H&E) or Verhoeff von Gieson (VVG) staining. The digital scans of whole section from each animal were generated with a ScanScope scanner and then visualized and analyzed using Aperio image view software. VVG-stained sections from each animal were evaluated for medial wall thickness of pulmonary arteries (50-250 μm diameter) in blinded manner. Wall thickness and outer diameter of approximately 25 muscularized arteries were measured in each section at two perpendicular points and then averaged. The percentage medial wall thickness was then calculated as described before . Immunohistochemistry staining of paraffin-embedded lung sections was performed as previously described  with primary antibodies including α-SMA, factor VIII, from Dako Corporation (Carpentaria, CA, USA), HIF-1α, from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and PDGF-BB, from Abcam, Inc. (Cambridge, MA).
Cell culture and treatments
Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin. Cells were treated with viral proteins: Tat 1-72 (1 μM, University of Kentucky), gp-120CM or gp-120LAV (100 ng/ml, Protein Sciences Corporation, Meriden, CT) for 24 hrs or 1 hr followed by western blot analysis and ROS quantification, respectively. Tat or gp120 stock solution was heat-inactivated by boiling for 30 min. For treatment with CCR5 neutralizing antibody or IgG isotype control (10 μg/ml, R&D systems) or; with antioxidant cocktail (0.2 mM ascorbate, 0.5 mM glutathione, and 3.5 μM α-tocopherol), cells were pre-treated with inhibitors for 30 min followed by treatment with gp-120 CM.
Quantification of cellular oxidative stress using dichlorofluorescein (DCF) assay
Pulmonary endothelial cells were treated with 5-(and -6)-carboxy-2', 7'-dichlorodihydroflourescein diacetate (DCFH-DA) (Molecular Probes, Inc.) for 30 min followed by treatment with viral protein(s) for 1 hr. In the presence of H2O2, DCFH is oxidized to a fluorescent DCF within the cytoplasm which was read by fluorescent plate reader at an excitation of 485 nm with an emission of 530 nm .
Transfection of pulmonary endothelial cells with small interfering (si) RNA
The silencer select pre-designed and validated siRNA duplexes targeting HIF-1α were obtained from Applied Biosystems (Carlsbad, CA). Cells were also transfected with silencer select negative control siRNA for comparison. HPMVECs were transfected with 10 nM siRNA using Hiperfect transfection reagent (Qiagen, Valencia, CA) as instructed by the manufacturer. The transfected cells were then treated with or without cocaine and/or Tat for 24 hrs followed by protein extraction for western blot analysis.
Real-Time RT-PCR analysis
We used Real-Time RT-PCR to analyze RNA extracted from frozen lungs of HIV-1 Tg rats and WT controls obtained after non-fixative perfusion. Quantitative analysis of HIF-1a, PDGF and ET-1 mRNA in Tg and WT rats was done using primers from SA Biosciences (Frederick, MD) by Real-Time RT-PCR using the SYBR Green detection method. Total RNA was isolated from frozen lung tissues by lysis in Trizol and was then converted into first strand cDNA to be used for real-time PCR. Detection was performed with an ABI Prism 7700 sequence detector. The average Ct value of the house-keeping gene, HPRT, was subtracted from that of target gene to give changes in Ct (dCt). The fold-change in gene expression (differences in dCt, or ddCt) was then determined as log2 relative units.
Western Blot Analysis
Frozen rat lung tissues or endothelial cells were lysed in lysing buffer (Sigma, St. Louis, MO) containing protease inhibitors. Protein estimation in these samples was measured using the micro-BCA method protein assay kit (Pierce Chemical Co., Rockford, IL). Western blot analyses were performed using primary antibodies against HIF-1α (Santa Cruz), PDGF-BB (Santa Cruz), and β-actin (Sigma). The secondary antibodies used were horseradish peroxidase-conjugated anti-mouse or anti-rabbit (1:5000, Pierce Chemical Co) and detection was performed using the enhanced chemiluminescence system (Pierce Chemical Co.). The NIH imageJ software was used for densitometric analysis of immunoblots.
Statistical analysis was performed using two-way analysis of variance with a post-hoc Student t- test or non-parametric Wilcoxon Rank-Sum test as appropriate. To test for association of RV/LV+septum ratio with other mediators, the non-parametric Spearman's rank-sum correlation coefficient was used and coefficient of determination (R2) was calculated. Exact two-sided p-values were calculated for all the analysis, using SAS 9.1 software (SAS Institute, Inc., Cary, NC, USA). A type I error rate of 5% was used for determining statistical significance.
Pulmonary vascular remodeling in HIV-Tg rats
Characterization of pulmonary vascular lesions in HIV-Tg rats
Right ventricular hypertrophy (RVH) in HIV-Tg rats
Increased expression of HIF-1a and PDGF-BB in HIV-Tg rats
Increased expression of PDGF-BB in HIV-protein(s) treated pulmonary microvascular endothelial cells
Reactive oxygen species are involved in HIV-protein mediated PDGF-BB induction
ROS dependent stimulation of HIF-1α is necessary for HIV-protein mediated PDGF-BB induction
Next to determine the involvement of HIF-1α in gp-120 mediated regulation of PDGF-BB expression, we used HIF-1α specific siRNA knock down experiments. First we optimized that the transfection of HPMVECs with 10nM HIF-1α siRNA was efficient in decreasing around 80% of gp-120CM induced HIF-1α expression when compared to cells transfected with non-specific siRNA control (Figure 9B). Furthermore, the HIF-1α siRNA transfected cells showed significant decrease in the expression of PDGF-BB in the presence of gp-120CM when compared with untransfected or non-specfic siRNA transfected gp-120CM treated cells (Figure 9C) thus underscoring the role of HIF-1α activation in the gp-120CM mediated PDGF-BB expression in pulmonary endothelial cells.
In this study, we offer histological and physiologic evidence of pulmonary vascular remodeling with significant thickening of medial layer of the arteries, elevated RV mass in the non-infectious rat model of HIV-1. Pulmonary arteriopathy exhibited by the HIV-Tg rats was manifested primarily by smooth muscle proliferation within the medial wall, endothelial disruption with little indication of endothelial cell proliferation but absence of classic plexiform lesions. Although RV hypertrophy in the HIV-Tg rats is suggestive of concomitant right heart pressure overload, the presence of pulmonary arteriopathy alone does not necessarily predict pulmonary hypertension. Furthermore, in humans only a fraction of individuals with HIV develop PAH, suggesting that the etiology of HIV-PAH is multi-factorial and complex where multiple insults such as HIV infection, drugs of abuse, and genetic predilection may be necessary to induce clinical disease. Therefore, one could hypothesize that the viral protein(s) provides the first 'hit' and second 'hit' such as administration of stimulants may lead to more severe pathology in these HIV-Tg rats.
Inflammation is considered to play an important role in HIV- associated pulmonary vascular remodeling with accumulation of macrophages and T lymphocytes found in the vicinity of pulmonary vessels of pulmonary hypertension patients [46, 47]. Consistent with these findings we also observed infiltration of mononuclear cells near or around the thickened vessels with mild interstitial pneumonitis as described before in this model . HIV-1 infection is known to stimulate monocyte/macrophages and lymphocytes to secrete elevated levels of cytokines, growth factors and viral proteins such as Nef, Tat and gp-120, [10–16] that can then initiate endothelial injury, SMC proliferation and migration, leading to the development of HIV-PAH [8–10, 18, 26, 48]. It is plausible that the medial wall thickening, an important determinant of pulmonary vascular resistance, discovered in HIV-Tg rat model is the result of integrated effects of various HIV proteins and the related inflammatory mediators including PDGF-BB.
Examination of the HIV-1 Tg rat lungs revealed increased staining of PDGF-BB in macrophages around hypertrophied vessels and in endothelial cells. Earlier studies suggest induction of PDGF-BB by endothelial cells  but not by SMCs  in response to hypoxia. Furthermore, the vasculature and lungs of this HIV-Tg rat model has earlier been demonstrated to be under significant oxidative stress [32, 33]. Along the lines, we observed enhanced expression of HIF-1α, a crucial transcription factor responsible for sensing and responding to oxidant stress and hypoxic conditions , suggesting, in part, the involvement of ROS/HIF-1α pathway in the over expression of PDGF-BB. HIF-1α controls a large program of genes critical to the development of pulmonary arterial hypertension [29, 31, 52, 53]. Interestingly, the expression of HIF-1α and PDGF was not only elevated and positively associated with each other in the lungs of the HIV-1 Tg rats, but the quantity of each was directly related, in a linear fashion, to the degree of increase in the right ventricular hypertrophy (RV/LV+septum ratio).
While these correlations in the HIV-1 transgenic model are consistent with our hypothesis, our in-vitro work in pulmonary endothelial cells validates that viral protein mediated oxidative stress/HIF-1α pathway results in induction of PDGF-BB. The injury to the endothelium, an initiating event in PAH  is known to be associated with the induction of oxidative stress . HIV-associated proteins, Tat and gp-120, as confirmed by our findings and others, demonstrate the ability to invoke oxidative stress mediated endothelial dysfunction [27, 28, 44, 55]. In addition, results demonstrating enhanced levels of HIF-1α in viral protein treated pulmonary endothelial cells, are in concert with the previous findings supporting the activation and accumulation of HIF-1α by HIV-1 through the production of ROS . PDGF-BB, known to be involved in hypoxia-induced vascular remodeling, [30, 57, 58]) has been suggested to be up-regulated in a HIF-dependent manner, but the mechanism by which HIF-1α and PDGF levels are elevated during vascular remodeling associated with PAH, are still not completely understood. A putative HIF-response element on PDGF-gene has been identified  but the studies demonstrating the direct involvement of HIF-1α in the regulation of PDGF expression are lacking. Here, we provide evidence validating the significance of HIF-1α in the pathogenesis of HIV-associated vascular dysfunction, and report the novel finding that its response to viral protein generated oxidative stress is to augment PDGF expression in the pulmonary endothelium. To our knowledge, this is the first report validating that HIV-1 viral proteins through the activation of HIF-1α induce PDGF expression.
The HIV-1 virus is unable to actively infect endothelial cells due to the absence of necessary CD4 receptors. However, viral proteins have been demonstrated to act on endothelial cells through direct binding to their CCR5 (R5) or CXCR4 (X4) co-receptors . This is corroborated with our findings showing mitigation of gp-120 CM response to increase PDGF-BB expression in the presence of CCR5 neutralizing antibody. Maximum PDGF-BB expression and ROS production was seen on treatment with R5-type gp-120 that is expected to be secreted in abundance by infiltrated HIV-infected CCR5+ T cells  and macrophages seen around the pulmonary vascular lesions associated with PAH . In addition, studies on co-receptor usage of HIV have shown that virus utilizing CCR5 as a co-receptor is the predominant type of virus found in HIV-infected individuals . Furthermore, R5gp120 has been reported earlier to induce the expression of cell-cycle and cell proliferation related genes more strongly than X4 gp-120 in peripheral blood mononuclear cells  and this differential potency of gp-120 effect may be present in pulmonary endothelial cells as well.
In summary, we demonstrate that the influence of HIV-1 proteins alone, without viral infection, is associated with pulmonary arteriopathy including accumulation of HIF-1α and PDGF as observed in the HIV-1 Tg rats. Furthermore, our in-vitro findings confirm that HIV-1 viral protein mediated generation of oxidative stress and resultant activation of HIF-1α leads to subsequent induction of PDGF expression in pulmonary endothelial cells. Consistent with a possible role of PDGF in the development of idiopathic PAH, the correlation of this mediator with RVH, does suggest this pathway may be one of the many insults involved in the development of HIV-related pulmonary arteriopathy and potentially HIV-PAH.
Supported by grants from Parker B. Francis Foundation, NIH 1R03DA031589, Joseph C. Cates Family Foundation, and Kansas IDDRC (P30 NICHD HD 02528). We are grateful to Jo Wick for her expert statistical assistance and Benjamin Ladner for his help in editing the manuscript.
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