2979The effect of proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors on endothelial progenitor cells

Abstract Background Endothelial progenitor cells (EPCs) have an important role in the process of vascular repair by promoting re-endothelialization following endothelial injury. We hypothesized that proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors, which reduce cardiovascular events, will increase the level of EPCs and thus affect the process of vascular repair. Therefore, we sought to investigate the effect of PCSK9 inhibitors on circulating EPCs. Methods Study population included patients with known stable CAD who were initiated PCSK9 inhibitors. Blood samples were drawn and evaluated for EPCs at baseline and after treatment (1 month). Circulating EPCs were then assessed quantitatively by the expression of VEGFR-2, CD34 and CD133 using flow cytometry, and functionally by the formation of colony forming units (CFUs). Results Our preliminary cohort included 12 patients (median age of 69 years), 31% of whom were female. At baseline, total cholesterol and low density lipoprotein levels were 190 (IQR 180, 227) mg/dL and 123 (IQR 107, 154) mg/dL, respectively. Following 1-month of therapy with a PCSK9 inhibitor and along with a decrease in LDL to a median of 58 (IQR 50, 67) mg/dL, we observed an increase in the expression of CD34(+)/VEGFR-2(+) (1.2% (IQR 0.6, 1.6) to 3.0% (IQR 1.2, 4.5), P=0.07) and CD133(+)/VEGFR-2(+) (0.8% (IQR 0.7, 1.4) to 1.7% (IQR 0.6, 4.0), P=0.5). Proliferation of EPCs was confirmed microscopically (1 CFUs (IQR 1, 1.5) to 1.5 CFUs (IQR 1.5, 2.5), P=0.016) (Figure 1) and by an MTT assay (0.16 (IQR 0.12, 0.19) to 0.19 (IQR 0.17, 0.21), p=0.016). Conclusions These preliminary results in patients with CAD demonstrate that treatment with PCSK9 inhibitors is associated with higher levels of EPCs, thus promote endothelial repair. This finding may represent a novel mechanism of action of PCSK9 inhibitors, which might have important future clinical implications. </jats:sec

Angiogenic Peptides Improve Blood Flow and Promote Capillary Growth in a Diabetic and Ischaemic Mouse Model

AbstractObjectivesIt is a common clinical observation that collateral vessel development is impaired in diabetic patients with ischaemic vascular diseases. Consequently, alternative revascularisation strategies in diabetic patients are needed. This study presents the effect and mechanism of new peptide therapeutic angiogenesis in an ischaemic and diabetic mouse model.DesignStreptozocin-injected mice that had undergone hind-limb ischaemia were treated with angiogenic peptides. Blood flow restoration was calculated by laser Doppler imager and corroborated by histological section. For the mechanism study, endothelial cells were exposed to hypoxia and high glucose concentrations to study the effect of the peptides on proliferation and anti-apoptosis.ResultsThe peptides significantly restored blood perfusion 21 days after surgery in the diabetic mice (p < 0.01) by neo-vascularisation, corroborated by an increase in capillary density. In addition, the peptides induced the proliferation of hypoxic endothelial cells (p < 0.01) and protected the cells from apoptosis in high glucose cultures.ConclusionsThis is the first approach for treatment of ischaemic vascular disease with peptides in a diabetic mouse model

Quantitative and functional evaluation of endothelial progenitor cells in patients with cardiac amyloidosis

Abstract Background Endothelial microvascular dysfunction is a known mechanism of injury in cardiac amyloidosis (CA), but evidence regarding the level and function of endothelial progenitor cells (EPCs) in patients with CA is lacking. Methods Study population included patients with light-chain or transthyretin (ATTR) CA. Patients with diagnosed heart failure and preserved ejection fraction (HFpEF) without monoclonal gammopathy and a 99mTc-DPD scan incompatible with TTR were used as controls. Blood circulating EPCs were assessed quantitatively by the expression of VEGFR-2(+), CD34(+) and CD133(+) using flow cytometry, and functionally by the formation of colony forming units (CFUs). MTT assay was used to demonstrate cell viability. Tests were repeated 3 months following the initiation of amyloid-suppressive therapies (either ATTR-stabilizer or targeted chemotherapy) in CA patients. Results Our preliminary cohort included 14 CA patients (median age 74 years, 62% ATTR CA). Patients with CA vs. patients with HFpEF (n=8) demonstrated lower expression of CD34(+)/VEGFR-2(+) cells [0.51% (IQR 0.4, 0.7) vs. 1.03% (IQR 0.6, 1.4), P=0.043] and CD133(+)/VEGFR-2(+) cells [0.35% (IQR 0.23, 0.52) to 1.07% (IQR 0.6, 1.5), P=0.003]. Functionally, no differences were noted between groups. Following the initiation of amyloid-suppressive therapies in CA patients, we observed the up-regulation of CD34(+)/VEGFR-2(+) cells [2.47% (IQR 2.1, 2.7), P&amp;lt;0.001] and CD133(+)/VEGFR-2(+) cells [1.38% (IQR 1.1, 1.7), P=0.003]. Moreover, functionally, active EPCs were evident microscopically by their ability to form colonies (from 0.5 CFUs [IQR 0, 1.5) to 2 CFUs (IQR 1, 3.5), P=0.023]. EPCs' viability was demonstrated by an MTT assay [0.12 (IQR 0.04, 0.12) to 0.24 (IQR 0.16, 0.3), p=0.014]. Conclusions These preliminary results demonstrate reduced EPCs levels in CA patients indicating significant microvascular impairment. Amyloid-targeted therapies induce the activation of EPCs, thus possibly promoting endothelial regeneration. These findings may represent a novel mechanism of action of amyloid-suppressive therapies EPCs in CA patients and during therapy Funding Acknowledgement Type of funding source: None </jats:sec

Pathways Mediating the Interaction between Endothelial Progenitor Cells (EPCs) and Platelets

INTRODUCTION: Endothelial progenitor cells (EPCs) have an important role in the process of vascular injury repair. Platelets have been shown to mediate EPC recruitment, maturation and differentiation. Yet, the mechanism underlying this interaction is unclear. We, therefore, aimed to examine whether direct contact between platelets and EPCs is essential for the positive platelets-EPC effect, and to investigate the main mediators responsible for the improvement in EPCs function. METHODS: Human EPCs were isolated from donated buffy coats and cultured in either: 1. EPCs co-incubated with platelets placed in a 1 µm-Boyden chamber. 2. EPCs incubated with or without platelets in the presence or absence of bFGF/PDGF Receptor inhibitor (PDGFRI). After 7 days culture, EPCs ability to form colonies, proliferate and differentiate was examined. Culture supernatants were collected and growth factors levels were evaluated using ELISA. Growth factors mRNA levels in EPCs were evaluated using RT-PCR. RESULTS AND CONCLUSIONS: After 7 days culture, EPCs functional properties were higher following co-incubation with platelets (directly or indirectly), implying that direct contact is not essential for the platelet's positive effect on EPCs. This effect was reduced by PDGFRI inhibition. Additionally, higher levels of PDGFB in EPCs-platelets supernatant and higher levels of PDGFC mRNA in EPCs co-incubated with platelets were found. In contrast, FGF and other potential mediators that were examined and inhibited did not significantly affect the interaction between platelets and EPCs. Thus, we conclude that PDGF has a central role in the interaction between platelets and EPCs. Further study is required to examine additional aspects of EPC-platelets interaction