Eleonora Petito

In vivo platelet activation and platelet hyperreactivity in abacavir-treated HIV-infected patients

Abacavir (ABC) has been associated with ischaemic cardiovascular events in HIV-infected patients, but the pathogenic mechanisms are unknown. Aim of our study was to assess whether ABC induces in vivo platelet activation and ex vivo platelet hyper-reactivity. In a retrospective, case-control study, in vivo platelet activation markers were measured in 69 HIV-infected patients, before starting therapy and after 6-12 months of either ABC (n=35) or tenofovir (TDF) (n=34), and compared with those from 20 untreated HIV-infected patients. A subgroup of patients was restudied after 28-34 months for ex vivo platelet reactivity. In vivo platelet activation markers were assessed by ELISA or flow cytometry, ex vivo platelet reactivity by light transmission aggregometry (LTA) and PFA-100®. Thein vitro effects of the ABC metabolite, carbovir triphosphate, on aggregation and intra-platelet cGMP were also studied. sPLA2, sPsel and sGPV increased significantly 6-12 months after the beginning of ABC, but not of TDF or of no treatment. Ex vivo platelet function studies showed enhanced LTA, shorter PFA-100® C/ADP closure time and enhanced platelet expression of P-sel and CD40L in the ABC group. The intake of ABC blunted the increase of intraplatelet cGMP induced by nitric oxide (NO) and acutely enhanced collagen-induced aggregation. Preincubation of control platelets with carbovir triphosphate in vitro enhanced platelet aggregation and blunted NO-induced cGMP elevation. In conclusion, treatment with ABC enhances in vivo platelet activation and induces platelet hyperreactivity by blunting the inhibitory effects of NO on platelets. These effects may lead to an increase of ischaemic cardiovascular events.

Impact of Tenofovir Versus Abacavir on HIV-Related Endothelial Dysfunction

Ischemic cardiovascular events are a frequent complication of long-lasting HIV infection and have been attributed either to the infection itself or to antiretroviral therapy (ART). Some cohort studies suggested that in particular abacavir may be associated with an increased cardiovascular risk, but the pathogenic mechanisms remain unknown. Recently, abacavir use was significantly associated with incident cardiovascular disease in HIV-infected veterans and it was related to higher risk of acute myocardial infarction (AMI). Endothelial dysfunction is a central mechanism in atherosclerosis and a marker of cardiovascular risk. We have previously shown that HIVinfected patients present an endothelial dysfunction that tends to improve upon ART, suggesting that the infection itself rather than therapy is responsible for endothelial damage. In the present study we compared treatment with abacavir (ABC) and tenofovir (TDF) for their impact on endothelial dysfunction. In a retrospective, case-control study, plasma levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and monocyte chemoattractant protein-1 (MCP-1), two markers of endothelial dysfunction, were measured by flow cytometry using a bead-based assay (FlowCytomix, Bender MedSystems, Vienna, Austria) in 69 HIV-infected patients, before starting therapy and after 6–12 months of either ABC (n = 35, 18 females, age 44 – 12 years) or TDF (n = 34, 17 females, age 43 – 11 years). The two groups were similar for baseline immune-virologic status and all patients showed an increase of CD4 + count and a lowering of viral load after therapy, without differences between ABC and TDF. Twenty-one patients were recalled 28–34 months after the beginning of therapy (11 ABC and 10 TDF) for the measurement of peripheral vascular endothelial function by finger arterial pulse wave amplitude with the Endo-PAT2000 system, and of circulating endothelial cells (CECs) by flow cytometry. Two control groups were included: 20 HIV-infected patients (6 females, age 42 – 9 years), from whom blood samples were collected at diagnosis and after 6–12 months without therapy, and 10 healthy ageand gender-matched controls. All results are expressed as means – standard error of the mean (SEM). Differences between groups were assessed by using one-way analysis of variance (ANOVA) with the Kruskal-Wallis post hoc test, using the GraphPad Prism version 4.00 for Windows software (GraphPad, San Diego, CA). At diagnosis endothelial activation markers were significantly higher in HIV-infected patients than in healthy controls (sVCAM-1: 783 – 157 versus 518 – 53 ng/mL, p < 0.05; MCP-1: 293 – 22 versus 228 – 20 pg/mL, p < 0.05). After 6–12 months of treatment, sVCAM-1 and MCP-1 decreased significantly in the TDF group, although not to normal levels, but not in the ABC group, while in untreated patients endothelial dysfunction did not change, or even slightly worsened, at followup (Fig. 1A and B). Upon reevaluation after 28–34 months of treatment endothelial function, as assessed by peripheral arterial tonometry and CECs, was still significantly altered in HIV-infected patients as compared with healthy controls (Fig. 1C), with ABC-treated patients showing significantly higher CECs than controls and tendentially higher than TDF-treated patients (Fig. 1D); moreover, in ABC-treated but not in TDFtreated patients, CECs inversely correlated with endothelial function (Fig. 1E and F). Endothelial dysfunction detected by impaired peripheral arterial tonometry and increased CECs, two parameters not previously measured in HIV-infected patients, is a reliable marker of vascular damage and predicts late cardiovascular ischemic events. Therefore, our data confirm that chronic HIV-infection impairs endothelial function and show that antiretroviral treatment with TDF, but less with ABC, improves it. Very recent data showing that TDF selectively decreases the production of cardiovascular disease-related inflammatory cytokines are supportive of our conclusions. Whether persistent endothelial dysfunction in ABC-treated patients contributes to the suggested increased cardiovascular risk associated with ABC use needs to be assessed in prospective studies.

Potential anti-inflammatory effects of maraviroc in HIV-positive patients: A pilot study of inflammation, endothelial dysfunction, and coagulation markers

Abstract Persistent immune activation and chronic inflammation significantly contribute to non-AIDS morbidity in HIV-infected patients. The HIV inhibitor maraviroc (MVC) targets the cellular chemokine CCR5 HIV co-receptor, which is involved in important inflammatory pathways. MVC could have significant anti-inflammatory and anti-atherosclerotic effects, also reducing immune activation. We designed a pilot study to determine which plasma biomarkers of inflammation, endothelial dysfunction, and hypercoagulability were modified by MVC in 2 groups of 10 patients starting MVC-free or MVC-containing regimens. Ten age- and gender-matched healthy controls were also included. We found higher levels of all inflammatory biomarkers in HIV-infected patients compared to healthy controls. Both groups showed decreasing levels of interleukin (IL)-17, IL-10, and macrophage inflammatory protein (MIP)-1a following the achievement of viral suppression. Vascular cell adhesion molecule (VCAM)-1 levels were decreased in the MVC group and increased in the MVC-free group. In conclusion, some inflammatory biomarkers tend to decrease with the salvage regimen; MVC was not associated with a better impact on these measured markers.

The Migration of Platelets and their Interaction with Other Migrating Cells

Platelets, beyond their well-described role in haemostasis and thrombosis, act as inflammatory cells playing an active role in several inflammatory conditions. As observed with other inflammatory cells platelets can migrate in vitro, either randomly or in the direction of a chemotactic agent, and in vivo, into inflammed tissues in response to different stimuli. In this chapter we will summarize the current knowledge about the mechanisms that regulate platelet chemotaxis, the evidence for the ability of platelets to migrate in vitro and in vivo, and the mechanisms by which platelets influence chemotaxis of other cells.

Platelets Contribute to the Accumulation of Matrix Metalloproteinase Type 2 in Synovial Fluid in Osteoarthritis

Inflammation plays a role in the initiation and progression of osteoarthritis (OA), a chronic degenerative joint disorder. Platelets are inflammatory cells, contain and release matrix metalloproteinases (MMPs) and favour the release of these enzymes, key effectors of cartilage and subchondral bone degradation, by other cells; however, their role in OA has not been investigated yet. Our aims were (1) to assess the presence of platelets and of MMP-2 in synovial fluid (SF) of OA patients; (2) to evaluate the contribution of platelets to MMP-2 release by fibroblast-like synoviocytes (FLS); and (3) to investigate if hyaluronic acid (HA) interferes with these processes. SF was collected from 27 OA patients before and after treatment with intra-articular HA (20 mg/2 mL). Moreover, FLS were co-cultured with platelets, and the release of MMP-2 in supernatants was measured. Our results show that platelets are present in OA SF and show markers of activation. OA SF also contains relevant amounts of MMP-2. Co-incubation of platelets with FLS favours the release of MMP-2 by the interaction of platelet surface P-selectin with FLS CD44 by a mechanism involving the activation of pAkt and pSrc in FLS. Administration of HA to OA patients decreased the infiltration of platelets in SF and reduced the levels of MMP-2. The addition of HA in vitro inhibited the release of MMP-2 by FLS triggered by the interaction with platelets. In conclusion, our data show that platelets may contribute to joint degeneration in OA by favouring the accumulation of MMP-2 in SF.

A dichotomy in platelet activation: Evidence of different functional platelet responses to inflammatory versus haemostatic stimuli

INTRODUCTION Platelets participate in inflammatory disorders through a variety of different functional responses, including chemotaxis, platelet-leukocyte complex formation and facilitation of leukocyte recruitment that are thought to be distinct from platelet aggregation. This may account for why classical anti-platelet drugs have failed to ameliorate inflammatory disorders where platelets are known to participate, suggesting that distinct pathways may control inflammatory and haemostatic functions of platelets. In the present study, we have therefore investigated the effect of different stimuli on several different functions of platelets preferentially involved either in haemostasis or in inflammation. MATERIALS AND METHODS Human platelets were stimulated with either inflammatory (fMLP, histamine, IL-1β, LPS, MDC/CCL22, SDF-1α/CXCL12 and 5-HT) or haemostatic (ADP, collagen, convulxin, epinephrine, TRAP-6 and U46619) stimuli. Aggregation, platelet-leukocyte complex formation, platelet migration and platelet protein phosphorylation were assessed. RESULTS Haemostatic stimuli induced platelet aggregation, whilst inflammatory agonists induced platelet migration. The haemostatic stimuli, with the exception of epinephrine, and some of the inflammatory stimuli induced platelet-leukocyte complex formation, even if to a different extent. Furthermore, inflammatory stimuli induced a shorter lasting profile of platelet protein phosphorylation compared with haemostatic stimuli. CONCLUSIONS Stimulation of platelets with inflammatory stimuli triggers the activation of non haemostatic functions different from those induced by haemostatic stimuli, supporting the existence of alternative platelet responses depending on the stimulus (haemostatic or inflammatory). A deeper understanding of the biochemical pathways behind these functional differences may lead to the development of novel therapeutic options targeting the inflammatory actions of platelets, without affecting their critical role in haemostasis.

THU0194 Role of Platelets in the Pathogenesis of Osteoarthritis and Biological Effects of Hyaluronic Acid: in Vivo and in Vitro Study

Background Although osteoarthritis (OA) is a degenerative joint disorder, it has been demonstrated that inflammation takes part in the development and progression of this condition. Matrix metalloproteinases (MMPs), including MMP-2, are involved in the cartilage breakdown observed in OA. Platelets (PLTs) are involved in different inflammatory conditions, including rheumatoid arthritis, contain and release several MMPs, including MMP-2, and enhance MMPs expression by other cell types. However, their role in OA has not been investigated yet. Hyaluronic acid (HA) is a normal component of extracellular matrix and synovial fluid (SF). Intra-articular (IA) administration of HA in OA is widely employed in clinical practice with good clinical efficacy. Objectives Aims of our study were to investigate the role of platelets and MMP-2 in the pathogenesis of OA, to test the biologic activity of IA-HA in OA patients and to analyze the effects of HA on the interaction between platelets and synoviocytes in vitro. Methods Thirty-eight patients with knee OA were screened and twenty-four patients were included in the study. Systemic therapy with non-steroidal anti-inflammatory drugs or corticosteroids and previous IA corticosteroid treatment represented exclusion criteria. Five injections of IA sodium hyaluronate (500-730 KDa) were administrated weekly. SF samples were collected prior to the first (T0) and at the last (T1) injection. Total cell and PLT count, platelet-leukocyte aggregates (PLT activation marker) and MMP-2 levels were measured in SF. For clinical assessment at T0, T1 and 3 months after the last injection (T3) the Western Ontario and Mc Masters University (WOMAC) scale and VAS for knee pain were employed. Fibroblast-like synoviocytes (FLS) were isolated from untreated OA-SF and cultured with PLTs in presence or absence of HA at different concentrations for 24 hours. Platelet P-selectin was measured by flow-cytometry and MMP-2 in culture supernatants was quantified by zymography. Results Total cell count in SF was significantly reduced after IA-HA (p<0.05). PLT count, platelet-leukocyte aggregates and MMP-2 concentration were significantly decreased at T1 (all p<0.05). WOMAC scale and pain VAS were reduced at T1 (p<0.0001) and this decrease was maintained at T3. MMP-2 production by FLS was significantly higher in the presence of PLTs and this increase was significantly reduced by co-incubation with HA. P-selectin expression, marker of PLT activation, was significantly higher in PLTs cultured with FLS and P-selectin blockade reduced MMP-2 release in culture supernatants. Conclusions Our results show that PLTs, in addition to other inflammatory cells, participate in the pathogenesis of OA by the induction of MMP-2 release by FLS possibly via P-selectin, thus contributing to cartilage breakdown. Treatment with HA decreases count and activation of PLT and levels of MMP-2 in the SF. PLT and synoviocytes interaction leads to platelet activation and MMP-2 release enhancement. Hence, PLTs may represent a new therapeutic target in OA. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4585

Role of platelet activation in the cardiovascular complications associated with HIV infection: differential effect of abacavir versus tenofovir

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK