Key Points Fibrosis in the liver is a common cause of liver disease, partially mediated by platelet TGF-β1 as shown in a mouse model of liver injury. Depleting platelet TGF-β1 results in decreased liver fibrosis suggesting that blocking platelet TGF-β1 may ameliorate or prevent fibrosis.
Cardiovascular mechanical stresses trigger physiological and pathological cellular reactions including secretion of Transforming Growth Factor β1 ubiquitously in a latent form (LTGF-β1). While complex shear stresses can activate LTGF-β1, the mechanisms underlying LTGF-β1 activation remain unclear. We hypothesized that different types of shear stress differentially activate LTGF-β1. We designed a custom-built cone-and-plate device to generate steady shear (SS) forces, which are physiologic, or oscillatory shear (OSS) forces characteristic of pathologic states, by abruptly changing rotation directions. We then measured LTGF-β1 activation in platelet releasates. We modeled and measured flow profile changes between SS and OSS by computational fluid dynamics (CFD) simulations. We found a spike in shear rate during abrupt changes in rotation direction. OSS activated TGF-β1 levels significantly more than SS at all shear rates. OSS altered oxidation of free thiols to form more high molecular weight protein complex(es) than SS, a potential mechanism of shear-dependent LTGF-β1 activation. Increasing viscosity in platelet releasates produced higher shear stress and higher LTGF-β1 activation. OSS-generated active TGF-β1 stimulated higher pSmad2 signaling and endothelial to mesenchymal transition (EndoMT)-related genes PAI-1, collagen, and periostin expression in endothelial cells. Overall, our data suggest variable TGF-β1 activation and signaling occurs with competing blood flow patterns in the vasculature to generate complex shear stress, which activates higher levels of TGF-β1 to drive vascular remodeling.
Combination antiretroviral therapies (cART) have markedly reduced mortality in HIV infection. However, cardiovascular disease (CVD), including heart failure linked to fibrosis, remains a major cause of morbidity and mortality in HIV/cART patients. The magnitude of this risk increases with use of certain protease inhibitors (PI), but the underlying mechanism remains unclear. We showed that the PI ritonavir leads to increased plasma levels of the pro-fibrotic cytokine TGF-β1, cardiac dysfunction, and pathologic cardiac fibrosis in wild-type (wt) C57BL/6 mice. Mice with targeted depletion of platelet TGF-β1 had reduced cardiac fibrosis and partially preserved cardiac function following ritonavir exposure (Laurence, et al. PLoS One 2017;12:e0187185). Several groups have examined the effects of a variety of cART agents on agonist-induced platelet aggregation, but correlations with clinical CVD are weak. Since platelets are a rich source of TGF-β1, we hypothesized that ritonavir and other PIs linked clinically to an increased CVD risk directly activate platelets to release TGF-β1 and activate latent (L)TGF-β1 to initiate signaling for organ fibrosis. We examined the impact of clinically relevant doses of ritonavir, alone and in combination with two other contemporary PIs, atazanavir and darunavir, which are currently used along with low dose ritonavir in so-called PI-boosted cART regimens. We incubated human platelet-rich plasma and washed platelets with PIs alone or in combinations at various doses for 10 min at 37°C in a platelet aggregometer (BioData. Corp). Total and active TGF-β1 levels were measured by ELISA. For in vivo assessment, we treated wt mice with a low dose of ritonavir, as used in PI-boosted cART, and measured the levels of plasma TGF-β1 by ELISA, and TGF-β1 signaling in tissues by immunofluorescence imaging for pSmad2. We found that ritonavir dose-dependently increased total TGF-β1 release from freshly-isolated platelet-rich plasma and washed human platelets. This release was blocked by ceefurin-1 and MK517, potent inhibitors of the ATP binding cassette transporter ABCC4. Darunavir alone did not cause release of TGF-β1, and did not alter significantly ritonavir-induced TGF-β1 release (Figure-1A). Atazanavir alone did induce release of TGF-β1 from platelets and did not affect the extent of such release induced by ritonavir (Figure-1A). Since total TGF-β1 released from platelets must be activated in order to signal, we tested whether these PIs could activate LTGF-β1. Ritonavir alone, in low dose, activated TGF-β1 by 4-5-fold (Fig-1B). Darunavir alone did not activate LTGF-β1, and had only a minor effect on ritonavir-induced TGF-β1 activation (Fig-1B). In marked contrast, while atazanavir also did not activate LTGF-β1, it significantly inhibited ritonavir-induced LTGF-β1 activation (Fig-1B). For in vivo assessment, wt mice were injected daily for 8 weeks with ritonavir, which dose-dependently increased plasma TGF-β1 levels (mean levels with vehicle 2.1 ng/ml; 6.4 ng/ml with 5 mg/kg ritonavir; 8.5 ng/ml with 10 mg/kg ritonavir). Increased TGF-β1 levels correlated with development of pathologic fibrosis and increased phosphorylated Smad signaling in hearts of ritonavir-treated vs. vehicle-treated mice. Clinical correlations with these in vitro and in vivo mouse studies are important. The fact that ritonavir effected both release and activation of platelet TGF-β1 is consistent with its ability to induce cardiac fibrosis and dysfunction in mice, and its association with accelerated CVD in HIV-infected individuals. Our findings that low dose ritonavir in combination with darunavir induced release and activation of platelet TGF-β1, whereas atazanavir blocked TGF-β1 activation, are consistent with the strong association of ritonavir-boosted darunavir, but not ritonavir-boosted atazanavir, with CVD in the setting of HIV (Ryom, et al. Lancet-HIV 2018;5:e291-e300). Future work will examine the effects of other contemporary cART agents, including cobicistat, which is currently replacing ritonavir in many PI-boosted therapies and some integrase-boosted regimens, on TGF-β1 release and activation, for which correlations with clinical CVD are not yet available. Identification of the mechanism of pathologic fibrosis in the heart, and potentially other organs affected by certain cART regimens, such as the kidney, may suggest specific therapeutic interventions. Disclosures No relevant conflicts of interest to declare.
Although survival and quality of life have improved in patients with advanced heart failure (HF) after implantation of left ventricular assist devices (LVADs), they still pose risks of hemocompatibility-related complications, including thrombosis and bleeding. Development of biomarkers predictive of these LVAD-associated complications could guide decision making for both clinicians and patients. Recently, we showed higher plasma TGF-β1 levels within one-week after implantation with a miniaturized mechanical-bearing axial-flow pump HeartMate II (HM-II), and reasoned that platelet activation by the rotor may have caused the release of TGF-β1 in plasma in HF patients (Mancini et al. Transl. Res. 2018; 192:15-29). Recent clinical trials with the newest LVAD, the Heartmate 3 (HM-3), which uses a fully magnetically-levitated pump, showed superior clinical outcomes, including significantly reduced incidences of pump thrombosis and stroke (Mehra et al. N Engl J Med. 2019; 380:1618-1627). In this study, we evaluated release of TGF-β1 in plasma following implantation of HM-II and HM-3 LVADs compared to either coronary artery bypass graft (CABG) surgery or extracorporeal membrane oxygenation (ECMO) therapy. We measured serial total TGF-β1 levels in 38 Stage-D HF patients (11 received HM-II and 27 received HM-3). As a control, we collected blood samples from 10 patients undergoing CABG surgery, and 10 patients receiving ECMO therapy following acute onset cardio-pulmonary failure. Blood samples were collected before and 4-8 hours after procedures, and thereafter daily for up to one week. Plasma was prepared by centrifuging blood at 12,000 rpm for 5 min at 4°C within 10 min of blood drawing, which reduces in vitro release of TGF-β1 from platelets and thus allows accurate measurement of plasma TGF-β1. Total TGF-β1 levels were measured after acidification and neutralization of samples using DUO-ELISA kit (R&D Systems). Baseline total plasma TGF-β1 levels were higher in HF patients before LVAD implantation than in healthy controls [4.7 ± 1.9 ng/mL in HF patients (n= 38); 3.3 ± 0.8 ng/mL in healthy controls (n= 6); p=0.006)]. Total TGF-β1 levels surged transiently to 14.6 ± 6.1 ng/mL within 4-8 hours after LVAD implantation [(p<0.0001 compared to patients 4-12 hours after CABG surgery (3.6 ± 1.4 ng/mL) or ECMO therapy (4.9 ± 1.3 ng/mL)]. Interestingly, however, we found that the transient surge of TGF-β1 in HM-3 recipients was significantly lower than in HM-II recipients (Figure-1; p=0.04). TGF-β1 levels then gradually decreased and reached near basal levels 2-3 days after LVAD implantation, but remained significantly elevated in plasma of HM-II recipients until day 5 (p=0.049). TGF-β1 levels remained unchanged in both CABG and ECMO patients at all time points (Figure 1). We conclude that LVAD implantation causes a transient surge in total plasma TGF-β1 within a few hours after the procedure, presumably due to platelet activation by LVAD, not the surgery itself, as CABG or circulating blood through ECMO did not cause the surge. The observation that a reduced initial surge and lower levels of TGF-β1 in HM-3 vs. HM-II recipients needs further investigation to determine whether these differences are due to LVAD-specific factors (different rotors causing variable shear effects) or to confounding differences in implantation procedures, such as, by-pass time, cardiac tissue injury, number of platelet transfusions, blood suction with catheters etc. or other unknown factors. Our data suggest that serial TGF-β1 measurements after LVAD implantation may serve as a surrogate biomarker for platelet activation in association with hemocompatibility-related adverse events (Uriel et al. Circ. 2017; 135:2003-2012). Disclosures No relevant conflicts of interest to declare.
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