HIV protease inhibitors induce senescence and alter osteoblastic potential of human bone marrow mesenchymal stem cells: beneficial effect of pravastatin

HIV‐infected patients receiving antiretroviral therapy present an increased prevalence of age‐related comorbidities, including osteoporosis. HIV protease inhibitors (PIs) have been suspected to participate to bone loss, but the mechanisms involved are unknown. In endothelial cells, some PIs have been shown to induce the accumulation of farnesylated prelamin‐A, a biomarker of cell aging leading to cell senescence. Herein, we hypothesized that these PIs could induce premature aging of osteoblast precursors, human bone marrow mesenchymal stem cells (MSCs), and affect their capacity to differentiate into osteoblasts. Senescence was studied in proliferating human MSCs after a 30‐day exposure to atazanavir and lopinavir with or without ritonavir. When compared to untreated cells, PI‐treated MSCs had a reduced proliferative capacity that worsened with increasing passages. PI treatment led to increased oxidative stress and expression of senescence markers, including prelamin‐A. Pravastatin, which blocks prelamin‐A farnesylation, prevented PI‐induced senescence and oxidative stress, while treatment with antioxidants partly reversed these effects. Moreover, senescent MSCs presented a decreased osteoblastic potential, which was restored by pravastatin treatment. Because age‐related bone loss is associated with increased bone marrow fat, we also evaluated the capacity of PI‐treated MSCs to differentiate into adipocyte. We observed an altered adipocyte differentiation in PI‐treated MSCs that was reverted by pravastatin. We have shown that some PIs alter osteoblast formation by affecting their differentiation potential in association with altered senescence in MSCs, with a beneficial effect of statin. These data corroborate the clinical observations and allow new insight into pathophysiological mechanisms of PI‐induced bone loss in HIV‐infected patients.     

HIV protease inhibitor-specific alterations in human adipocyte differentiation and metabolism

Objective: Human immunodeficiency virus (HIV) patients on antiretroviral regimens frequently develop a syndrome of abnormal fat distribution, insulin resistance, and dyslipidemia. This lipodystrophic syndrome has been most closely linked to the use of HIV protease inhibitors (PIs). Several mechanisms have been postulated to explain these adverse effects of PIs, based largely on studies of rodent adipocytes. Intriguingly, atazanavir, a newer PI equally effective against HIV, is associated with fewer signs of lipodystrophy. We hypothesized that the less deleterious clinical effects of atazanavir would be reflected in physiological differences observed in PI‐treated adipocytes. Research Methods and Procedures: We compared the effects of atazanavir and an older PI associated with lipodystrophy, ritonavir, on differentiation, gene expression, adipocytokine secretion, and insulin signaling in a human adipocyte cell line. Results: Ritonavir inhibited human adipocyte differentiation and induced apoptosis to a greater extent than atazanavir. Treatment of mature adipocytes with ritonavir, but not atazanavir, also selectively decreased insulin signaling. Moreover, ritonavir also selectively decreased expression of adiponectin, an insulin‐sensitizing adipocytokine, while inducing interleukin‐6, a proinflammatory cytokine implicated in insulin resistance. Discussion: These data suggest that the distinct metabolic side effect profiles of these PIs could be a consequence of their differential effects on adipocyte physiology.     

Central memory CD4+ T cell responses in chronic HIV infection are not restored by antiretroviral therapy

A strong CD4(+) T cell response has been correlated with better control of HIV infection. However, the effect of HIV on the maintenance of Ag-specific memory CD4(+) T cells is not fully understood. We characterized the function and phenotype of memory CD4(+) T cells generated by mumps and influenza A or B viruses in HIV-infected individuals receiving highly active antiretroviral therapy (n = 21), HIV-infected long-term nonprogressors (n = 10), and HIV-seronegative volunteers (n = 10). We observed significantly decreased proliferation of the Ag-specific central memory CD4(+) T cell population (CD28(+)/CCR7(+)/CD45RA(-)) in the antiretroviral treated HIV-infected individuals compared with the seronegative controls. Restored CD4(+) T cell count and decreased HIV viral load while on highly active antiretroviral therapy did not result in increased proliferation, whereas nadir CD4(+) T cell count predicted the presence of Ag-specific proliferation. Our results indicate that HIV infection leads to impaired maintenance of virus-induced or vaccine-generated central memory CD4(+) T cells that is not restored by HAART.     

Visceral adiposity,C-peptide levels,and low lipase activities predict HIV-dyslipidemia

Protease inhibitor-based highly active antiretroviral therapy (PI-HAART) has been implicated in dyslipidemia, peripheral insulin resistance, and abnormal adipose tissue deposition in human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome, or AIDS. In vitro evidence indicates that some PIs reduce adipocyte lipoprotein (LPL) and hepatic lipase (HL) expression and activities. We examined whether LPL and HL activities are reduced in HIV-infected patients with dyslipidemia. Fasting serum lipids, glucoregulatory hormones, and postheparin LPL and HL activities, as well as whole body and regional adiposity, were measured in 19 HIV-seronegative controls, 9 HIV+ patients naive to all anti-HIV medications, 9 HIV+ patients naive to PIs, 9 HIV+ patients with prior PI experience but not currently receiving PIs, and 47 HIV+ patients receiving PI-HAART. The PI-HAART group had low LPL and HL activities. However, multiple linear regression analysis indicated that low postheparin LPL activity contributed only partially to HIV-dyslipidemia. Central adiposity and high C-peptide levels (an indicator of high insulin secretion) were stronger predictors of HIV-dyslipidemia. Low LPL and HL activities, by themselves, were insufficient to explain HIV-dyslipidemia because the PI-naive group had low LPL and HL activities but had normal adiposity, C-peptide levels, and serum lipid and lipoprotein levels. HDL-cholesterol was lower in PI-HAART and PI-naive groups than seronegative controls and was directly associated with LPL activity. These findings suggest that HIV-dyslipidemia is mediated primarily by factors that influence triglyceride and lipoprotein synthesis (e.g., central adiposity and hyperinsulinemia) and mediated only partially by factors that influence triglyceride clearance (e.g., lipase activity).     

Clinician gender is more important than gender concordance in quality of HIV care

Background: Previous studies have examined the impact of physician gender and gender concordance on preventive care, satisfaction, and communication. Less is known about how physician gender and gender concordance affect care for chronic illnesses, including HIV.Objective: This study sought to determine whether patient-clinician gender concordance (patient and clinician are of the same gender) influences receipt of protease inhibitor (PI) therapy and ratings of care among HIV-infected patients.Methods: We reviewed data from 1860 patients and 397 clinicians in the HIV Cost and Services Utilization Study, a nationally representative the association between gender concordance and time to first PI use, and multivariable logistic regression was utilized to examine the association of gender concordance with patients' problems with care and their overall rating of care.Results: Patients who had a male clinician received PIs earlier than those who had a female clinician (adjusted time ratio = 0.69 for having a male vs having a female clinician; P ≤ 0.01). Gender concordance was not a significant predictor of time to PI use. Gender discordance was associated with problems with feeling respected by clinicians. Female patients with a male clinician were most likely, and female patients with female clinicians were least likely, to report a problem with being treated with respect (P ≤ 0.01 for the interaction term). Gender discordance was not associated with other problems with care or with overall ratings of care.Conclusions: Gender discordance was associated with perceived problems of being treated with respect by clinicians, but not with time to receipt of PIs, overall ratings of care, coordination of care, or obtaining information. The perception of not being respected may represent a significant barrier to care that is particularly worse for women, in that most HIV-infected women receive their care from male clinicians.     

The frequency of malaria is similar among women receiving either lopinavir/ritonavir or nevirapine-based antiretroviral treatment

HIV protease inhibitors (PIs) show antimalarial activity in vitro and in animals. Whether this translates into a clinical benefit in HIV-infected patients residing in malaria-endemic regions is unknown. We studied the incidence of malaria, as defined by blood smear positivity or a positive Plasmodium falciparum histidine-rich protein 2 antigen test, among 444 HIV-infected women initiating antiretroviral treatment (ART) in the OCTANE trial (A5208; ClinicalTrials.gov: NCT00089505). Participants were randomized to treatment with PI-containing vs. PI-sparing ART, and were followed prospectively for ≥48 weeks; 73% also received cotrimoxazole prophylaxis. PI-containing treatment was not associated with protection against malaria in this study population.     

Mechanisms of lipid elevations associated with the treatment of patients with HIV infection

OBJECTIVE: Dyslipidemia and other metabolic abnormalities, which are associated with the use of highly active antiretroviral therapy (HAART) for the treatment of HIV infection, are of concern to patients and healthcare providers. The objective of this review is to present the current understanding of the dyslipidemia associated with the protease inhibitor (PI)-component of HAART: its possible origin, potential consequences, and management techniques. DATA SOURCES, STUDY SELECTION: Peer-reviewed, published literature was identified via MEDLINE. Other sources included abstracts from recent HIV-related conferences that presented data pertinent to the topic. Studies were selected based on their impact on our understanding of HIV infection and its treatment. DATA EXTRACTION, SYNTHESIS: Relevant portions of the publications considered were compiled and conclusions were drawn based on the clinical experience of the author. CONCLUSION: Dyslipidemia associated with current PIs should be a serious consideration when initiating long-term treatment of HIV infection. Current management techniques that include lipid-lowering agents may be improved and streamlined by incorporating a lipid-friendly PI into HAART.     

Recurrent pneumonia mortality risk in a HIV/AIDS Puerto Rican cohort.

Recurrent pneumonia (RP) within 12 months is one of the AIDS diagnosis criteria. To gain knowledge of RP infection in HIV-infected patients, we studied 145 RP cases detected in a cohort of 2,996 HIV patients in Puerto Rico between Jan. 1992-Dec. 2001. The RP prevalence was 4.8%; 77.2% were males and 62.1% were injecting drug users (IDU). At the time of RP diagnosis, the mean CD4+ T cell count was 93.8 cells/mm3, 59.3% were in antiretroviral treatment, 13% had received the pneumococcal vaccine and 84.8% had another AIDS related condition. Over 37% received two or more antiretroviral medications. The death rate in the first year after the RP diagnosis was 63.4%. A Cox proportional hazard analysis showed that CD4+ T cells <200/mm3 (p<0.05), history of toxoplasmosis (p<0.01), wasting syndrome (p<0.01), esophageal candidiasis (p<0.05) and lower number of antiretroviral medications (p<0.05) increased their mortality risk. The studied patients had a highly compromised immune system and a very low pneumococcal vaccination percent at the time of RP diagnosis. Low CD4+ T cells significantly increased the hazard and mortality risk of the cases studied. Antecedents of antiretroviral therapy in these patients ensure a better outcome with lower mortality. Efforts to increase the vaccination rate should reduce the RP incidence in our HIV-infected population.     

D-Dimer Levels before HIV Seroconversion Remain Elevated Even after Viral Suppression and Are Associated with an Increased Risk of Non-AIDS Events

The mechanism underlying the excess risk of non-AIDS diseases among HIV infected people is unclear. HIV associated inflammation/hypercoagulability likely plays a role. While antiretroviral therapy (ART) may return this process to pre-HIV levels, this has not been directly demonstrated. We analyzed data/specimens on 249 HIV+ participants from the US Military HIV Natural History Study, a prospective, multicenter observational cohort of >5600 active duty military personnel and beneficiaries living with HIV. We used stored blood specimens to measure D-dimer and Interleukin-6 (IL-6) at three time points: pre-HIV seroconversion, ≥6 months post-HIV seroconversion but prior to ART initiation, and ≥6 months post-ART with documented HIV viral suppression on two successive evaluations. We evaluated the changes in biomarker levels between time points, and the association between these biomarker changes and future non-AIDS events. During a median follow-up of 3.7 years, there were 28 incident non-AIDS diseases. At ART initiation, the median CD4 count was 361cells/mm³; median duration of documented HIV infection 392 days; median time on ART was 354 days. Adjusted mean percent increase in D-dimer levels from pre-seroconversion to post-ART was 75.1% (95% confidence interval 24.6–148.0, p = 0.002). This increase in D-dimer was associated with a significant 22% increase risk of future non-AIDS events (p = 0.03). Changes in IL-6 levels across time points were small and not associated with future non-AIDS events. In conclusion, ART initiation and HIV viral suppression does not eliminate HIV associated elevation in D-dimer levels. This residual pathology is associated with an increased risk of future non-AIDS diseases.     

Inhibition of P-Glycoprotein by HIV Protease Inhibitors Increases Intracellular Accumulation of Berberine in Murine and Human Macrophages

Background

HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages.

Methodology and Principal Findings

Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.

Conclusion and Significance

HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.     

Advanced HIV Disease at Enrolment in HIV Care: Trends and Associated Factors over a Ten Year Period in Cambodia

Background

Early HIV diagnosis and enrolment in care is needed to achieve early antiretroviral treatment (ART) initiation. Studies on HIV disease stage at enrolment in care from Asian countries are limited. We evaluated trends in and factors associated with late HIV disease presentation over a ten-year period in the largest ART center in Cambodia.

Methods

We conducted a retrospective analysis of program data including all ARV-naïve adults (> 18 years old) enrolling into HIV care from March 2003-December 2013 in a non-governmental hospital in Phnom Penh, Cambodia. We calculated the proportion presenting with advanced stage HIV disease (WHO clinical stage IV or CD4 cell count <100 cells/μL) and the probability of ART initiation by six months after enrolment. Factors associated with late presentation were determined using multivariate logistic regression.

Results

From 2003–2013, a total of 5642 HIV-infected patients enrolled in HIV care. The proportion of late presenters decreased from 67% in 2003 to 44% in 2009 and 41% in 2013; a temporary increase to 52% occurred in 2011 coinciding with logistical/budgetary constraints at the national program level. Median CD4 counts increased from 32 cells/μL (IQR 11–127) in 2003 to 239 cells/μL (IQR 63–291) in 2013. Older age and male sex were associated with late presentation across the ten-year period. The probability of ART initiation by six months after enrolment increased from 22.6% in 2003–2006 to 79.9% in 2011–2013.

Conclusion

Although a gradual improvement was observed over time, a large proportion of patients still enroll late, particularly older or male patients. Interventions to achieve early HIV testing and efficient linkage to care are warranted.     

The Cost-Effectiveness of Directly Observed Highly-Active Antiretroviral Therapy in the Third Trimester in HIV-Infected Pregnant Women

Background

In HIV-infected pregnant women, viral suppression prevents mother-to-child HIV transmission. Directly observed highly-active antiretroviral therapy (HAART) enhances virological suppression, and could prevent transmission. Our objective was to project the effectiveness and cost-effectiveness of directly observed administration of antiretroviral drugs in pregnancy.

Methods and Findings

A mathematical model was created to simulate cohorts of one million asymptomatic HIV-infected pregnant women on HAART, with women randomly assigned self-administered or directly observed antiretroviral therapy (DOT), or no HAART, in a series of Monte Carlo simulations. Our primary outcome was the quality-adjusted life expectancy in years (QALY) of infants born to HIV-infected women, with the rates of Caesarean section and HIV-transmission after DOT use as intermediate outcomes. Both self-administered HAART and DOT were associated with decreased costs and increased life-expectancy relative to no HAART. The use of DOT was associated with a relative risk of HIV transmission of 0.39 relative to conventional HAART; was highly cost-effective in the cohort as a whole (cost-utility ratio $14,233 per QALY); and was cost-saving in women whose viral loads on self-administered HAART would have exceeded 1000 copies/ml. Results were stable in wide-ranging sensitivity analyses, with directly observed therapy cost-saving or highly cost-effective in almost all cases.

Conclusions

Based on the best available data, programs that optimize adherence to HAART through direct observation in pregnancy have the potential to diminish mother-to-child HIV transmission in a highly cost-effective manner. Targeted use of DOT in pregnant women with high viral loads, who could otherwise receive self-administered HAART would be a cost-saving intervention. These projections should be tested with randomized clinical trials.     

Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue

Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation.     

Hematopoietic-stem-cell-based gene therapy for HIV disease

Although combination antiretroviral therapy can dramatically reduce the circulating viral load in those infected with HIV, replication-competent virus persists. To eliminate the need for indefinite treatment, there is growing interest in creating a functional HIV-resistant immune system through the use of gene-modified hematopoietic stem cells (HSCs). Proof of concept for this approach has been provided in the instance of an HIV-infected adult transplanted with allogeneic stem cells from a donor lacking the HIV coreceptor, CCR5. Here, we review this and other strategies for HSC-based gene therapy for HIV disease.     

Whole body leucine flux in HIV-infected patients treated with or without protease inhibitors

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of l-[1-(13)C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 +/- 0.09 vs. 0.81 +/- 0.07 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]leucine enrichment, P = 0.06; or 0.70 +/- 0.10 vs. 0.99 +/- 0.08 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI- groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 +/- 0.10 vs. 0.18 +/- 0.06 micromol x kg FFM x (-1) x min(-1) in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected.     

Iatrogenic Osteoporosis,Bilateral Hip Osteonecrosis,and Secondary Adrenal Suppression in an Hiv-Infected Man Receiving Inhaled Corticosteroids and Ritonavir-Boosted Highly Active Antiretroviral Therapy

ObjectiveTo report the first case of severe osteoporosis associated with a vertebral pathologic fracture and osteonecrosis of femoral heads in an HIV-infected man receiving inhaled corticosteroids and ritonavir-boosted antiretroviral therapy.MethodsWe describe an HIV-infected man with severe osteoporosis, bilateral hip osteonecrosis, and secondary adrenal suppression, including detailed clinical, laboratory, and radiographic data, and review the related literature.ResultsA 60-year-old man with a 15-year history of HIV infection and a medical history of long-standing bronchiectasis treated with inhaled corticosteroids and hypogonadism treated with testosterone was referred to the endocrinology clinic after experiencing an osteoporotic vertebral fracture. He was taking ritonavir-boosted antiretroviral therapy. Osteonecrosis of both hips was also diagnosed, which required total hip replacement therapy.Laboratory evaluation revealed adrenal insufficiency due to increased effect of exogenous inhaled steroids and no other secondary causes of osteoporosis. A bone densitometry study showed osteoporosis of both hips and the lumbar spine. He was treated with intravenous pamidronate. During treatment, he developed bilateral femoral fractures after minor trauma.ConclusionsGiven the potential for increased serum levels of inhaled corticosteroids in patients taking ritonavirboosted highly active antiretroviral therapy, attention must be paid to the risk of bone loss in HIV-infected patients taking inhaled corticosteroids. Prescribing calcium and vitamin D supplementation and considering early osteoporosis screening are reasonable measures for this patient population. Interaction between inhaled corticosteroids and ritonavir may increase risk of hypothalamus-pituitary-adrenal axis suppression. (Endocr Pract. 2011;17:74-78)     

Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence

Lipodystrophic syndromes associated with mutations in LMNA, encoding A-type lamins, and with HIV antiretroviral treatments share several clinical characteristics. Nuclear alterations and prelamin A accumulation have been reported in fibroblasts from patients with LMNA mutations and adipocytes exposed to protease inhibitors (PI). As genetically altered lamin A maturation also results in premature ageing syndromes with lipodystrophy, we studied prelamin A expression and senescence markers in cultured human fibroblasts bearing six different LMNA mutations or treated with PIs. As compared to control cells, fibroblasts with LMNA mutations or treated with PIs had nuclear shape abnormalities and reduced proliferative activity that worsened with increasing cellular passages. They exhibited prelamin A accumulation, increased oxidative stress, decreased expression of mitochondrial respiratory chain proteins and premature cellular senescence. Inhibition of prelamin A farnesylation prevented cellular senescence and oxidative stress. Adipose tissue samples from patients with LMNA mutations or treated with PIs also showed retention of prelamin A, overexpression of the cell cycle checkpoint inhibitor p16 and altered mitochondrial markers. Thus, both LMNA mutations and PI treatment result in accumulation of farnesylated prelamin A and oxidative stress that trigger premature cellular senescence. These alterations could participate in the pathophysiology of lipodystrophic syndromes and lead to premature ageing complications.