title: Heparin-based Analogues and The Control of Vascular Proliferation
Creators: name: Wang, Kai-Wen
Publisher: name: University of Liverpool Repository
Year: 2021

description: Stent insertion into disease-narrowed arteries often damages blood vessels and triggers vascular smooth muscle (VSM) proliferation as a healing response, re-blocking arteries. To combat this, anti-proliferative agents can be incorporated into stents, but these delay healing, inhibit endothelial cell (EC) re-growth and promote thrombosis. This study aims to identify novel heparin-based analogues that inhibit VSM proliferation, promote EC proliferation and retain anti-thrombotic activity. In MTT-assays, incubation with naive heparin complexed with either Na+, K+, Mg2+ or Ca2+ ions (10 μg/μl for 4 days in normal growth media, GM) significantly reduced human coronary artery smooth muscle cell (HCASMC) growth compared to incubation in GM alone (P&lt;0.01). The inhibitory effect ranged from 48 ± 4.5 % (Na-heparin) to 28 ± 13.7 % (Mg-heparin). Cation-complexed forms of normal heparin also reduced human coronary artery endothelial cell (HCAEC) number but to a lesser extent: 19 ± 0.3% (Na-heparin (P&lt;0.01)) to 5.0 ± 3.5% (Ca-heparin (not significant)). The ratio of HCAEC:HCASMC cell growth thus showed that Na-, K-, Mg- and Ca-heparin have the desired effect of suppressing HCASMC proliferation, while having minimal effect on HCAECs. Partially or fully-desulphated heparin analogues complexed with either Na+, K+, Mg2+ or Ca2+ also showed promising activity profiles; partially-desulphated heparin 4 (predominant repeating structure; I2SA6OHNAc) consistently being the best performing analogue across all cations. In transwell migration assays, cationic desulphated heparin analogues significantly delayed HCASMC migration (P&lt;0.01), but had no significant effect on HCAECs. These effects were not due to induction of apoptosis and, importantly, all cationic desulphated heparins retained their differential effects on HCASMC/HCAECs in dual culture systems. Heparins with appropriate activity profiles were screened for their effects on blood coagulation and ability to interact with platelet factor 4 (PF4), a key determinant of heparin-induced thrombocytopenia. As expected, heavily-desulphated cationic heparin analogues lost anti-coagulant activity. Native (non-denatured) gel electrophoresis followed by silver staining was used to visualise protein complexes formed through the interaction of heparin analogues and PF4. All cationic forms of heparin tested formed PF4/heparin complexes. Mechanistically, differential effects of heparin analogues likely result from differences in growth factor (GF) signalling. In RT-PCR screens, transcripts for platelet-derived growth factor receptors, PDGFRA/B, were expressed only in HCASMCs. By growing HCASMCs in different growth factors, however, we determined that signalling via PDGFRs is not the primary cause of the differential heparin effects. Here, the anti-proliferative effects of heparin analogues were maintained, or enhanced, in media containing only fibroblast growth factor (FGF2) or epidermal growth factor (EGF), while anti-proliferative activity was decreased in media supplemented with predominantly PDGF. To elucidate differential GF signalling, heparin analogue-treated HCASMC lysates were immunoblotted with anti-phospho-tyrosine antibodies. Here, although immunoreactive band ‘fingerprints’ associated with EGFR activation were identified, no clear banding pattern differences were observed in cells treated with different heparin analogues. Ultimately, more sophisticated proteomic analysis will be required, but the differential effects on HCASMCs/HCAECs likely represents differences in signalling downstream of receptor activation, with candidate pathways activated by FGFs and EGF. In conclusion, to the best of our knowledge this is the first comprehensive analysis of the effects of heparin compounds on human vascular cells. Results highlight partially-desulphated, Na-heparin 4 and Ca-heparin 4, as potential lead analogues with promising activity profiles that may ultimately form the basis for novel, next-generation stent coats.