<p> Calcium (Ca2+) is an important signaling molecule and hence its movement across cell membranes must be tightly regulated. The intracellular Ca2+ concentration ([Ca2+]i) in smooth muscle and endothelium controls the coronary tone. After stimulation, decreasing the [Ca2+]i back to resting levels is achieved mainly by the sodium calcium exchanger (NCX), the plasma membrane calcium pump (PMCA) or the sarcoendoplasmic reticulum calcium pump (SERCA). The present study will focus on NCX and its interactions with SERCA in the smooth muscle and endothelium of pig coronary artery.</p> <p> Aim 1 of my thesis is determination of activity levels of NCX in smooth muscle cells (SMC) and endothelial cells (EC). The NCX activity in cultured cells was approximately 5 times greater in EC than in SMC. The NCX inhibitors KB-R7943 and SEA 0400 blocked the NCX mediated Ca2+ entry, as did collapsing the Na+ gradient with monensin. NCX1 is the isoform largely responsible for NCX activity in SMC and EC. NCX activity was also assayed as the Ca2+ efflux in cultured cells and as Ca2+ uptake in plasma membrane vesicles isolated from freshly isolated smooth muscle.</p> <p> Aim 2 is to assess the existence of a functional NCX mediated Ca2+ entry linked to SERCA in SMC. In the absence of thapsigargin, BAPTA loading SMC increased the NCX mediated uptake. Thapsigargin did not affect the Ca2+ uptake in BAPTA loaded cells but it inhibited the Ca2+ uptake in cells that were not loaded with BAPTA. These data are consistent with a model in which SER acts as a sink for the NCX mediated Ca2+ entry. However, with BAPTA chelation and the resulting lower intracellular Ca2+, the need for SER to act as a sink is eliminated, and NCX is driven in full force. EC did not demonstrate a NCX-SERCA linkage.</p> <p> Arterial SMC and EC differ in their structure and function. The function of SMC is the generation of tone which is achieved by the Ca2+ dependent contractile filaments. Since these filaments are distributed throughout the cell, Ca2+ must be transported to and removed from deep within the cell. As a result, the SER may play a large role in Ca2+ regulation in the SMC. Furthermore, SMC also contain higher levels of high affinity Ca2+ pumps (SERCA and PMCA) and thus Ca2+ is more tightly regulated. Endothelial cells release nitric oxide in response to an increase in [Ca2+]i, which relaxes the smooth muscle. The endothelial nitric oxide sythase produces nitric oxide and is located adjacent to the PM in EC. The SER that removes Ca2+ from deep within the cell cytosol may play a small role in Ca2+ dependent modulation of the endothelial nitric oxide synthase activity. Based on the Western blot data, EC contain a greater amount of the high capacity NCX, thus the larger quantities of Ca2+ can be removed from the cell and the vicinity of endothelial nitric oxide synthase.</p> / Thesis / Master of Science (MSc)

No / T-type Ca2+ channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca2+ channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca2+ channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca2+ channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca2+ channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca2+ channel-mediated proliferation by H2S is independent of the channels’ sensitivity to H2S. / This work was supported by the British Heart Foundation (PG/11/84/29146).

No / T-type Ca2+ channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe2+ and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca2+ channels. Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca2+ channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC50 values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca2+ ]i in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015). Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca2+ channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca2+ channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca2+ channels.

No / Cardiovascular disease is the principal cause of death in patients with type 2 diabetes (T2DM). Exposure of the vasculature to metabolic disturbances leaves a persistent imprint on vascular walls, and specifically on smooth muscle cells (SMC) that favours their dysfunction and potentially underlies macrovascular complications of T2DM. Current diabetes therapies and continued development of newer treatments has led to the ability to achieve more efficient glycaemic control. There is also some evidence to suggest that some of these treatments may exert favourable pleiotropic effects, some of which may be at the level of SMC. However, emerging interest in epigenetic markers as determinants of vascular disease, and a putative link with diabetes, opens the possibility for new avenues to develop robust and specific new therapies. These will likely need to target cell-specific epigenetic changes such as effectors of DNA histone modifications that promote or inhibit gene transcription, and/or microRNAs capable of regulating entire cellular pathways through target gene repression. The growing epidemic of T2DM worldwide, and its attendant cardiovascular mortality, dictates a need for novel therapies and personalised approaches to ameliorate vascular complications in this vulnerable population.

Yes / Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFβ) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.

Yes / Background/purpose Coronary heart disease is the leading cause of morbidity in patients with type 2 diabetes mellitus (T2DM), frequently resulting in a requirement for coronary revascularization using the internal mammary artery (IMA) or saphenous vein (SV). Patency rates of SV grafts are inferior to IMA and further impaired by T2DM whilst IMA patencies appear similar in both populations. Smooth muscle cells (SMC) play a pivotal role in graft integration; we therefore examined the phenotype and proliferative function of IMA- and SV-SMC isolated from non-diabetic (ND) patients or those diagnosed with T2DM. Methods/materials SMC were cultured from fragments of SV or IMA. Morphology was analyzed under light microscopy (spread cell area measurements) and confocal microscopy (F-actin staining). Proliferation was analyzed by cell counting. Levels of RhoA mRNA, protein and activity were measured by real-time RT-PCR, western blotting and G-LISA respectively. Results IMA-SMC from T2DM and ND patients were indistinguishable in both morphology and function. By comparison, SV-SMC from T2DM patients exhibited significantly larger spread cell areas (1.5-fold increase, P < 0.05), truncated F-actin fibers and reduced proliferation (33% reduction, P < 0.05). Furthermore, lower expression and activity of RhoA were observed in SV-SMC of T2DM patients (37% reduction in expression, P < 0.05 and 43% reduction in activity, P < 0.01). Conclusions IMA-SMC appear impervious to phenotypic modulation by T2DM. In contrast, SV-SMC from T2DM patients exhibit phenotypic and functional changes accompanied by reduced RhoA activity. These aberrancies may be epigenetic in nature, compromising SMC plasticity and SV graft adaptation in T2DM patients.

Yes / Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus–smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus–smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs −112 and −106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus–smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR- 145 observed in type 2 diabetes mellitus–smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.

Yes / Abdominal aortic aneurysm (AAA) is a silent, progressive disease with high mortality and increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (5cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of differentiation marker miR-145 (2.2-fold, P<.001) and senescence marker SIRT-1 (1.3-fold, P<.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, P<.001 and 1.8-fold, P<.01 respectively, versus control cells) and displayed aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker H2AX (3.9-fold, P<.01 vs. control SMC). These features did not correlate with patients’ chronological age; and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at end-stage disease. Refinement of a porcine bioreactor model would facilitate study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression. / Supported in part by a grant from the Leeds Teaching Hospitals Charitable Foundation (9R11/8002)

Yes / Cardiovascular disease is a leading cause of morbidity and mortality. Smooth muscle cells (SMC) comprising the vascular wall can switch phenotypes from contractile to synthetic, which can promote the development of aberrant remodelling and intimal hyperplasia (IH). MicroRNA‐21 (miR‐21) is a short, non‐coding RNA that has been implicated in cardiovascular diseases including proliferative vascular disease and ischaemic heart disease. However, its involvement in the complex development of atherosclerosis has yet to be ascertained. Smooth muscle cells (SMC) were isolated from human saphenous veins (SV). miR‐21 was over‐expressed and the impact of this on morphology, proliferation, gene and protein expression related to synthetic SMC phenotypes monitored. Over‐expression of miR‐21 increased the spread cell area and proliferative capacity of SV‐SMC and expression of MMP‐1, whilst reducing RECK protein, indicating a switch to the synthetic phenotype. Furthermore, platelet‐derived growth factor BB (PDGF‐BB; a growth factor implicated in vasculoproliferative conditions) was able to induce miR‐21 expression via the PI3K and ERK signalling pathways. This study has revealed a mechanism whereby PDGF‐BB induces expression of miR‐21 in SV‐SMC, subsequently driving conversion to a synthetic SMC phenotype, propagating the development of IH. Thus, these signaling pathways may be attractive therapeutic targets to minimise progression of the disease. / King Saud University; College of Medicine , Riyadh, Saudi Arabia

No / Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K+ (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mmd-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF−1 (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mmd-glucose, since pinacidil-activated ATP-dependent K+ (KATP) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mmd-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.