Amy O’Brien-Ladner

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.

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.

Inhibition of RalA signaling pathway in treatment of non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and relatively resistant to chemotherapy. The most prevalent molecular abnormality in NSCLC is the overactivation of K-Ras proto-oncogene; therefore, elucidating down-stream Ras signaling in NSCLC is significantly important in developing novel therapies against this malignancy. Our work indicates that RalA, an important effector of Ras, is activated in NSCLC cell lines. While RalA was also overactivated in fetal human broncho-epithelial cells, RalBP1 (Ral binding protein-1), an important down-stream effector of RalA, was expressed at higher levels in cancer cell lines. Aurora kinase-A (AKA), an upstream activator of RalA, was also found to be active only in malignant cells. The outcome of inhibition of RalA (by gene specific silencing using a lentivirus) on the malignant phenotype of A549 cells was also studied. While proliferation and invasiveness of A549 cells were reduced upon silencing RalA, apoptosis and necrosis were elevated in such conditions. Additionally, the in vivo tumorigenesis of A549 cells was reduced upon partial inhibition of RalA and AKA using pharmacological inhibitors. Finally, we were interested in evaluating the level of active RalA in the fraction of NSCLC cells expressing cancer stem cell markers. For this purpose cells with increased expression of CD44 were separated from A549 cells and compared with cells with low level of expression of this marker and an unsorted population. A significant enhancement of RalA activation in high CD44+ cells was found as potential evidence for involvement of RalA signaling in initiation of the neoplastic procedure and an important contributor for tumor maintenance in NSCLC. Further studies can reveal therapeutic, preventive and diagnostic value of RalA pathway in this deadly disease.

Effects of hypoxia and nitric oxide on ferritin content of alveolar cells

Concentrations of ferritin in alveolar cells and on the alveolar surface are increased in patients with a variety of respiratory disorders. Ferritin synthesis by cells is modulated by iron content but is also influenced by stimuli other than iron. In this study we sought to determine whether in vitro exposure to hypoxia- or nitric oxide (NO)-induced ferritin accumulation or release by human alveolar macrophages (AMs) or a lung cancer-derived epithelial cell line (A549). Changes in cell content of iron and ferritin (L- and H-types), as well as ferritin content of cell supernatants, were determined after in vitro exposure to hypoxia (1% or 10% O(2), 18 hours) or the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.01-1.0 mmol/L, 18 hours). Exposure to 1% O(2) increased ferritin content in both cell types (>fourfold increase; P <.005) without changing iron content. Treatment with SNAP increased ferritin content of A549 cells in a dose-dependent manner, whereas treatment of AMs decreased cellular iron and ferritin content and increased supernate ferritin content. Pretreatment of cells with N-acetylcysteine (500 micromol/L) reduced hypoxia-induced ferritin accumulation in alveolar cells and completely inhibited NO-induced ferritin accumulation in A549 cells. These findings indicate that exposure to 1% O(2)can increase ferritin content in alveolar cells, whereas NO can increase ferritin content (A549 cells) or decrease ferritin content (AMs).

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.

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.

Hyperactive TGF-β 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-β super family of receptors. Here, now we demonstrate the effect of HIV-Tat and cocaine on the proliferative TGF-β signaling cascade. We observed a significant increase in the secretion of TGF-β1 ligand along with enhanced protein expression of TGFβ Receptor (TGFβR)-1, TGFβR-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 TGFβR-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 TGFβR cascades were found to contribute to hyper-proliferation. Finally the increase in the levels of phosphorylated TGFβR1 and TGFβR2 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.

Medical ICU admissions during weekday rounds are not associated with mortality: a single-center analysis.

This study investigated whether intensive care unit (ICU) admissions to the research team’s tertiary care academic hospital during morning rounds was associated with increased mortality. Discharge data were analyzed on 1912 patients admitted to the ICUs between July 2007 and June 2011. Measures included discharge disposition, time of admission to the ICU, source of admission, and expected mortality score. Descriptive statistics were generated to examine the proportion of subjects who died based on admission time to the ICU, and Pearson’s χ2 test was used to test the null hypothesis that mortality rates for admissions during rounds and those at other times of the day would be similar. No difference in mortality was detected between admissions during rounds and all other times, whether analyzed using a bivariate (P = .55) or multivariable (P = .78) analysis. In this study, mortality was associated with severity of illness and not associated with admission during morning rounds.