Caplice, Noel M.

Site specific targeting remains an elusive goal for stem cell and gene therapies in the cardiovascular field. One promising option involves use of modified 3D stent designs that deliver larger cell/gene payloads to specific disease sites using the versatility of percutaneous vascular access. Here, we engineered smooth muscle cells (SMCs) placed in a 3D metallic stent scaffold to deliver high local concentrations of an angiogenic gene (VEGF165) in a porcine model of chronic total occlusion involving ameroid placement on the proximal left circumflex (LCx) artery(n≥8 per group). Engineered SMCs were retained within the mesh stent structure (479 ± 47.4 SMC/mm2) and were competent for VEGF production in vitro (8.8 ± 1.8 ng/ml in media). Following implantation, micro CT analyses revealed that stents loaded with VEGF producing SMCs significantly enhanced vasa vasora microvessel density (5.43 ± 0.46 vs 2.14 ± 0.19 vessels/mm2, p<0.001) with a concomitant increase in tissue VEGF protein levels (277.9 ± 85.7 vs 18.8 ± 4.4 pg/mg protein, p<0.0001). Formation of endothelial cell colonies from peripheral blood mononuclear cells was significantly increased with the implantation of VEGF-secreting cells suggesting increased angiogenic potential (7.0 ± 2.0 versus 41.6 ± 4.5, EC:CFU p<0.0001). VEGF secreting mesh stent placement enhanced regional blood flow analyses determined by microsphere analyses as well as improving regional wall motion in the LCx-related territory (p<0.05) as well as left ventricular function assessed in terms of ejection fraction (34.2 ± 6.7% vs 61.2 ± 5.2% P<0.001) and dp/dt (p<0.001) Our data indicate robust clinically relevant angiogenesis can be achieved in a human scale porcine chronic vascular occlusion model following stent based delivery of angiogenic cells. This may have implications for percutaneous delivery of stem cells, genes and secreted therapeutic factors in cardiovascular disease in general and in creation of percutaneous microvascular bypass networks in the setting of chronic vaso-occlusive disease in particular.

The aim of this study was to characterise a recently identified adult smooth muscle stem-like/progenitor cell (SMSPC) and exploit the ability to differentiate contractile smooth muscle cells (SMC) from SMSPCs facilitating tissue engineering of small to medium arteries, an approach which is hampered by inadequate sources of, and scale up methodologies for autologous SMCs. We initially identified a novel SMSPC in the bone marrow (BM) of rats that retained classic stem cell-like characteristics (clonogenicity, unlimited self-renewal, high telomerase activity, expression of stem cell markers) whilst expressing low levels of SMC-like transcripts. Spontaneous differentiation favoured a SMC phenotype where ~20-fold increases in calponin and SM-MHC protein expression were observed 10 days post-embryoid body formation, an effect augmented by TGFβ (P < 0.0001). Differentiation of SMSPCs from an undifferentiated state towards contractile SMCs was Myocd-dependent. Overexpression of Myocd in SMSPCs increased their percentage contraction in response to AngII (P < 0.05) and KCL (P < 0.05) and Ca2+ signalling to levels indistinguishable from rat SMCs. Knockdown of SMC-specific repressors resulted in Myocd-dependent SMC differentiation of SMSPCs. Taking advantage of the fact that SMSPCs possessed unlimited self-renewal capacity ex vivo and could be successfully reprogrammed into functional SMCs, we determined the vascular tissue engineering potential of SMSPCs and showed these cells integrate as viable tissue engineered vascular grafts in vivo. Specifically, after 1 month implantation, a Myocd-GFP+/Isl1+ smooth muscle layer was observed in the grafts along with a vWF+ luminal endothelial layer and multi-layered collagen and elastin fibre formation throughout the graft wall. We have recently isolated cells with similar SMSPC phenotype and differentiation potential from human peripheral blood. This study demonstrates that adult SMSPCs derived from rat BM and human blood can be reprogrammed to efficiently generate large quantities of mature contractile SMCs, displaying great utility as a cellular backbone for tissue engineered vascular grafts, making them an attractive source for vascular cell therapy and surgical applications.

Background —Proliferation of the intima is an early lesion of saphenous vein graft disease. Early patency rates of radial artery grafts are acceptable, but little is known about their risk of intimal hyperplasia. Methods and Results —To develop a model of intimal hyperplasia, we incubated human saphenous veins, internal mammary arteries, and radial arteries (n=6, 8, and 10, respectively) in an organ culture with Rosewell Park Memorial Institute 1640 (30% serum) for 0, 4, 7, 10, and 14 days. Quantitative histological studies were performed, and the average intimal-to-medial (I/M) ratio was calculated for each incubation interval. After 10 and 14 days of culture, the I/M ratio increased in the saphenous veins ( P =0.03, P =0.04 versus 0 day, respectively). No significant increase occurred in the I/M ratio in either the internal mammary or radial arteries. Next, the ability of adenoviral gene transfers to inhibit intimal hyperplasia in the saphenous veins was evaluated. Adenoviral-mediated gene transfer of nitric oxide synthase significantly reduced the I/M ratio at 14 days compared with vehicle ( P =0.001) and virus ( P =0.004) controls. Conclusions —The human saphenous vein has a greater propensity for intimal hyperplasia than arterial grafts; the human radial artery behaves similarly to the internal mammary artery. In the future, gene therapy may augment nitric oxide synthase, limiting vein graft disease.

AbstractDisorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss.

Abstract —Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours ( P <0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively ( P <0.05), and these growth factors accounted for ≈50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.

Background —Plaque disruption and exposure of subendothelial procoagulants such as tissue factor (TF) to circulating factor VII/VIIa (FVII/VIIa) lead to intravascular thrombosis. Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of TF-induced coagulation that binds to factor Xa and the TF-FVIIa catalytic complex in a two-step process. The aim of this study was to determine the expression of TFPI within human atherosclerotic plaque and its role in modulation of TF activity. Methods and Results —We measured the level of TFPI antigen in human carotid plaque and determined the relationship between TFPI and TF activity within plaque. Furthermore, we examined the biological activity and immunolocalization patterns of TFPI within carotid plaque. TFPI was detectable (TFPI+ group) in 22 of 34 specimens (mean±SEM, 404.4±91.8 pg/mg) and undetectable (TFPI− group) in 12 of 34 specimens. In the TFPI− group, normalized TF activity was significantly greater than that in the TFPI+ group (0.28±0.04 vs 0.14±0.02 U/pg, P =0.002). Furthermore, neutralization of TFPI activity using a polyclonal antibody resulted in an 8-fold increase in TF activity in the TFPI+ group ( P =0.001) but had no effect in the TFPI− group. Immunostaining for TFPI showed localization to endothelial cells, vascular smooth muscle cells within the fibrous cap region of the plaque, and macrophages within the shoulder region of the plaque. Conclusions —Taken together, these data suggest that biologically active TFPI is present within human atherosclerotic plaque and is associated with attenuated TF activity.

Despite percutaneous intervention after an acute coronary syndrome, patients remain at high risk for recurrent events in the first year. Prior studies have shown that a plaque rupture can occur not only at a single culprit lesion site but also in other atherosclerotic plaques throughout the coronary vasculature in patients with stable angina, silent myocardial ischemia, and during acute coronary syndromes. A 71-year-old man who presented with exertional angina and who had a successful stent in a culprit right coronary artery is described in this article. After 4 weeks, he represented with accelerated angina. A prior lesion in the obtuse marginal artery, remote from the site of the index lesion, had progressed from a 30% to 90% stenosis. This case report demonstrates the systemic nature of acute coronary syndromes, highlights the inherent instability of coronary artery disease, and supports the notion of aggressive secondary prevention in these patients.