HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

BAckground:HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs). RESULTS: MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPAR\u3b3 activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. CONCLUSIONS: The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients

Study of HIV-1's pathogenesis in bone compartment: effects of conventional antiretroviral therapy and new approaches to eradication

L’obiettivo della tesi è studiare il virus HIV-1 in relazione alle alterazioni sistemiche, riscontrate nel paziente HIV-infetto, in particolare alterazioni a carico del sistema scheletrico, indotte dal virus o dall’azione dei farmaci utilizzati nella terapia antiretrovirale (HAART). L’incidenza dell’osteoporosi nei pazienti HIV-positivi è drammaticamente elevata rispetto alla popolazione sana. Studi clinici hanno evidenziato come alcuni farmaci, ad esempio inibitori della proteasi virale, portino alla compromissione dell’omeostasi ossea, con aumento del rischio fratturativo. Il nostro studio prevede un follow-up di 12 mesi dall’inizio della HAART in una coorte di pazienti naïve, monitorando diversi markers ossei. I risultati ottenuti mostrano un incremento dei markers metabolici del turnover osseo, confermando l’impatto della HAART sull’omeostasi ossea. Successivamente abbiamo focalizzato la nostra attenzione sugli osteoblasti, il citotipo che regola la sintesi di nuova matrice ossea. Gli esperimenti condotti sulla linea HOBIT mettono in evidenza come il trattamento, in particolare con inibitori della proteasi, porti ad apoptosi nel caso in cui vi sia una concentrazione di farmaco maggiore di quella fisiologica. Tuttavia, anche concentrazioni fisiologiche di farmaci possono regolare negativamente alcuni marker ossei, come ALP e osteocalcina. Infine esiste la problematica dell’eradicazione di HIV-1 dai reservoirs virali. La HAART riesce a controllare i livelli viremici, ciononostante diversi studi propongono alcuni citotipi come potenziali reservoir di infezione, vanificando l’effetto della terapia. Abbiamo, perciò, sviluppato un nuovo approccio molecolare all’eradicazione: sfruttare l’enzima virale integrasi per riconoscere in modo selettivo le sequenze LTR virali per colpire il virus integrato. Fondendo integrasi e l’endonucleasi FokI, abbiamo generato diversi cloni. Questi sono stati transfettati stabilmente in cellule Jurkat, suscettibili all’infezione. Una volta infettate, abbiamo ottenuto una significativa riduzione dei markers di infezione. Successivamente la transfezione nella linea linfoblastica 8E5/LAV, che porta integrata nel genoma una copia di HIV, ha dato risultati molto incoraggianti, come la forte riduzione del DNA virale integrato.The aim of this thesis is to study HIV-1 virus and its relation with systemic alterations observed in HIV-positive patients, in particular alterations of the skeletal system, induced by the virus or drugs, used in antiretroviral therapy (HAART). The osteoporosis frequency in HIV-positive patients is dramatically higher in respect to healthy people. Clinical studies showed how some drugs (e.g. viral protease’s inhibitors) lead to bone homeostasis impairment, increasing bone fractures’ risk. In our study, we followed up a cohort of naïve patients for 12 months, starting from the beginning of HAART, monitoring several bone markers. Our results evidenced an increase in bone turnover’s metabolic markers, confirming the impact of HAART on bone homeostasis. Later, we focused on osteoblasts, the cell type responsible of new bone matrix’s synthesis. The experiments directed on HOBIT cells revealed how the antiretroviral treatment, particularly protease inhibitors, causes apoptosis in case and higher than physiological drug concentration an had been used. However, even physiological drugs’ concentrations may downregulate bone markers, such as ALP and osteocalcin. Eventually, we have the question of HIV-1’s eradication from viral reservoirs. HAART can successfully control HIV’s viremia. Nevertheless, diverse cell types are thought to be potential reservoirs of infection, thwarting the effects of therapy. That is why we set up a new molecular approach to eradication: taking advantage of the integrase enzyme to specifically recognize the viral LTR sequences, toward hit the integrated provirus. Blending integrase and FokI endonuclease, we obtained various clones. These clones had been stably transfected into Jurkat cells, susceptible to infection. Once infected, we obtained a significant reduction in markers of infection. Thereafter the transfection of the lymphoblastoid cell line 8E5/LAV (which harbours an integrated copy of HIV into cell genome) returned very encouraging results, such as the strong reduction of integrated viral DNA