Grafts enriched with subamnion-cord-lining mesenchymal stem cell angiogenic spheroids induce post-ischemic myocardial revascularization and preserve cardiac function in failing rat hearts.

A crucial question in post-ischemic cell therapy refers to the ideal method of cell delivery to the heart. We hypothesized that epicardial implantation of subamnion-cord-lining mesenchymal stem cells (CL-MSC) angiogenic spheroids embedded within fibrin grafts (SASG) facilitates donor cell survival and enhances cardiac function in failing rat hearts. Furthermore, we compared the efficacy of this approach applied through two delivery methods. Spheroids made of 1.5×10(4) human CL-MSC coated with 2×10(3) human umbilical vein endothelial cells were self-assembled in hanging drops. SASG were constructed by embedding 150 spheroids in fibrin matrix. Except for untreated rats (MI, n=8), grafts were implanted 2 weeks after myocardial infarction upon confirmation of ensued heart failure through thoracotomy: SASG (n=8) and fibrin graft (FG, n=8); or video-assisted thoracoscopic surgery (VATS): SASG-VATS (n=8) and FG-VATS (n=7). In vivo CL-MSC survival was comparable between both SASG-treated groups throughout the study. SASG and SASG-VATS animals had decreased left ventricular end-diastolic pressure relative to untreated animals, and increased fractional shortening compared to MI and FG controls, 4 weeks after treatment. A 14.1% and 6.2% enhancement in ejection fraction from week 2 to 6 after injury was observed in SASG/SASG-VATS, paralleled by improvement in cardiac output. Treated hearts had smaller scar size, and more blood vessels than MI, while donor CL-MSC contributed to arteriogenesis within the graft and infarct areas. Taken together, our data suggest that SASG treatment has the potential to restore failing hearts by preserving cardiac function and inducing myocardial revascularization, while attenuating cardiac fibrosis. Furthermore, we introduce a method for minimally invasive in situ graft assembly.

Cord lining-mesenchymal stem cells graft supplemented with an omental flap induces myocardial revascularization and ameliorates cardiac dysfunction in a rat model of chronic ischemic heart failure.

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×10(6) CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p<0.05), contractility (+dP/dt, 3383.8±250.78 mmHg vs. 2464.9±191.8 mmHg, p<0.05), and the relaxation rate (-dP/dt, -2707.2±250.7 mmHg vs. 1948.7±207.8 mmHg, p<0.05), compared to MI control 6 weeks after ischemic injury. Furthermore, evidence of a 20.32% increase in the ejection fraction was observed in CL-MSCG+OM rats from week 2 to 6 after injury. Both CL-MSCG and CL-MSCG+OM led to an enhanced cardiac output (p<0.05), and attenuated the infarct size (35.7%±4.2% and 34.7%±4.8%), as compared to MI (60.7%±3.1%; p<0.01 and p<0.001, respectively). All treated groups had a higher arteriole density than controls. Yet, a higher amount of functional blood vessels, and a 20-fold increase in arteriole numbers were found in CL-MSCG+OM. Altogether, CL-MSCGs supplemented with vascular supply have the potential to repair the failing, chronically ischemic heart by improving myocardial revascularization, attenuating remodeling, and ameliorating cardiac dysfunction.

Post-ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid-enriched myocardial artificial grafts improves heart function in a rat model.

Angiogenesis plays a key role in post-ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)-enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP-luciferase-expressing rat cardiomyoblasts and enriched with 5 µm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG-treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham-operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post-implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end-systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p < 0.05; LV end-diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p < 0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p < 0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p < 0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven-fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p < 0.0001). Implantation of AA-enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function.

The role of R-spondin2 in keratinocyte proliferation and epidermal thickening in keloid scarring.

Keloids are found only in humans and the underlying biochemical mechanisms of their pathogenesis remain unknown. R-spondins (Rspos) are a relatively new group of secreted proteins known to be Wnt/ß-catenin signaling agonists, but their role in keloids has yet to be elucidated. We investigated the expression levels of R-spondin2 (Rspo2) in cell lysates and conditioned media of monocultures and co-cultures of fibroblasts and keratinocytes derived from keloids and normal skin. In this study we found increased protein expression and secretion of Rspo2 in respective monocultures of keloid fibroblasts and keratinocytes when compared with their normal counterparts. Double-chamber co-culture experiments implicated the role of keloid keratinocytes (KKs) in the induction of Rspo2 secretion from fibroblasts because of epithelial-mesenchymal interactions. Addition of recombinant human Rspo2 in culture increased the proliferation of keratinocytes and it acted synergistically with Wnt3a through the canonical Wnt/ß-catenin pathway. Overexpression of Rspo2 in normal fibroblasts brought about thicker epidermis when compared with control fibroblasts in a skin organotypic culture model. This observation coincides with the hyperproliferative phenotype of thickened epidermis seen in keloids. Taken together, the results suggest the possible double paracrine action of KKs in inducing higher expression of Rspo2 in fibroblasts that promotes keratinocyte proliferation and epidermal thickening.