Amanda J. LeBlanc

Adipose-Derived Cell Construct Stabilizes Heart Function and Increases Microvascular Perfusion in an Established Infarct

We have previously shown that myocardial infarction (MI) immediately treated with an epicardial construct containing stromal vascular fraction (SVF) from adipose tissue preserved microvascular function and left ventricle contractile mechanisms. In order to evaluate a more clinically relevant condition, we investigated the cardiac recovery potential of an SVF construct implanted onto an established infarct. SVF cells were isolated from rat adipose tissue, plated on Vicryl, and cultured for 14 days. Fischer‐344 rats were separated into MI groups: (a) 6‐week MI (MI), (b) 6‐week MI treated with an SVF construct at 2 weeks (MI SVF), (c) 6‐week MI with Vicryl construct at 2 weeks (MI Vicryl), and (d) MI 2wk (time point of intervention). Emax, an indicator of systolic performance and contractile function, was lower in the MI and MI Vicryl versus MI SVF. Positron emission tomography imaging (18F‐fluorodeoxyglucose) revealed a decreased percentage of relative infarct volume in the MI SVF versus MI and MI Vicryl. Total vessel count and percentage of perfusion assessed via immunohistochemistry were both increased in the infarct region of MI SVF versus MI and MI Vicryl. Overall cardiac function, percentage of relative infarct, and percentage of perfusion were similar between MI SVF and MI 2wk; however, total vessel count increased after SVF treatment. These data suggest that SVF treatment of an established infarct stabilizes the heart at the time point of intervention by preventing a worsening of cardiac performance and infarcted volume, and is associated with increased microvessel perfusion in the area of established infarct.

Microvascular Repair: Post-Angiogenesis Vascular Dynamics

Please cite this paper as: LeBlanc AJ, Krishnan L, Sullivan CJ, Williams SK, Hoying JB. Microvascular repair: post‐angiogenesis vascular dynamics. Microcirculation19: 676–695, 2012.

Adipose-derived stromal vascular fraction cells isolated from old animals exhibit reduced capacity to support the formation of microvascular networks

UNLABELLED Adipose-derived regenerative and stem cells, defined collectively as the stromal vascular fraction (SVF), support the formation of neovascular networks at the site of implantation. The effect of advancing age on SVF cell population effectiveness towards stimulated neovascularization was evaluated. METHODS SVF was enzymatically isolated from adipose of young (ySVF, 4 months) or old (oSVF, 24 months) Fisher-344 rats, combined with type I collagen and polymerized. Encapsulated SVF was implanted subcutaneously into young Rag1 mice for two or four weeks. Angiogenic function of age-dependent SVF was also extensively evaluated in vitro using standard assays. RESULTS In vitro studies indicated no difference in angiogenic function between ySVF and oSVF (viability, proliferation, migration, and tube-formation). At two weeks post-implantation, there was no age-related difference in percent apoptosis in explanted constructs. By four weeks post-implantation, oSVF implants displayed 36% less total vessels/mm(2), 43% less perfused vessels/mm(2), and exhibited greater percent apoptosis compared to ySVF (n ≥ 12). Blocking thrombospondin-1 (Thbs-1), a protein found to be highly expressed in oSVF but not ySVF, increased the percent of perfused vascular volume and vessel diameters in oSVF constructs after two weeks compared to oSVF implants treated with control antibody. CONCLUSIONS Advancing donor age reduces the potential of adipose-derived SVF to derive a mature microcirculation, but does not hinder initial angiogenesis. However, modulation of Thbs-1 may improve this outcome. This data suggests that greater pruning, dysfunctional structural adaptation and/or poor maturation with initiation of blood flow may occur in oSVF.

Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue

The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms.

Systemically Delivered Adipose Stromal Vascular Fraction Cells Disseminate to Peripheral Artery Walls and Reduce Vasomotor Tone Through a CD11b+ Cell-Dependent Mechanism

Vasoactivity, an important aspect of tissue healing, is often compromised in disease and tissue injury. Dysfunction in the smaller vasoactive arteries is most impactful, given the role of these vessels in controlling downstream tissue perfusion. The adipose stromal vascular fraction (SVF) is a mix of homeostatic cells shown to promote tissue healing. Our objective was to test the hypothesis that autologous SVF cells therapeutically modulate peripheral artery vasoactivity in syngeneic mouse models of small artery function. Analysis of vasoactivity of saphenous arteries isolated from normal mice 1 week after intravenous injection of freshly isolated SVF cells revealed that pressure‐dependent artery vasomotor tone was decreased by the SVF cell isolate, but not one depleted of CD11b+ cells. Scavenging hydrogen peroxide in the vessel wall abrogated the artery relaxation promoted by the SVF cell isolate. Consistent with a CD11b+ cell being the relevant cell type, SVF‐derived F4/80‐positive macrophages were present within the adventitia of the artery wall coincident with vasorelaxation. In a model of artery inflammation mimicking a common disease condition inducing vasoactive dysfunction, the SVF cells potentiated relaxation of saphenous arteries without structurally remodeling the artery via a CD11b+ cell‐dependent manner. Our findings demonstrate that freshly isolated, adipose SVF cells promote vasomotor relaxation in vasoactive arteries via a hydrogen peroxide‐dependent mechanism that required CD11b+ cells (most likely macrophages). Given the significant impact of small artery dysfunction in disease, we predict that the intravenous delivery of this therapeutic cell preparation would significantly improve tissue perfusion, particularly in diseases with diffuse vascular involvement.

Physiological levels of thrombospondin-1 decrease NO-dependent vasodilation in coronary microvessels from aged rats.

Aging and cardiovascular disease are associated with the loss of nitric oxide (NO) signaling and a decline in the ability to increase coronary blood flow reserve (CFR). Thrombospondin-1 (Thbs-1), through binding of CD47, has been shown to limit NO-dependent vasodilation in peripheral vascular beds via formation of superoxide (O2 (-)). The present study tests the hypothesis that, similar to the peripheral vasculature, blocking CD47 will improve NO-mediated vasoreactivity in coronary arterioles from aged individuals, resulting in improved CFR. Isolated coronary arterioles from young (4 mo) or old (24 mo) female Fischer 344 rats were challenged with the NO donor, DEA-NONO-ate (1 × 10(-7) to 1 × 10(-4) M), and vessel relaxation and O2 (-) production was measured before and after Thbs-1, αCD47, and/or Tempol and catalase exposure. In vivo CFR was determined in anesthetized rats (1-3% isoflurane-balance O2) via injected microspheres following control IgG or αCD47 treatment (45 min). Isolated coronary arterioles from young and old rats relax similarly to exogenous NO, but addition of 2.2 nM Thbs-1 inhibited NO-mediated vasodilation by 24% in old rats, whereas young vessels were unaffected. Thbs-1 increased O2 (-) production in coronary arterioles from rats of both ages, but this was exaggerated in old rats. The addition of CD47 blocking antibody completely restored NO-dependent vasodilation in isolated arterioles from aged rats and attenuated O2 (-) production. Furthermore, αCD47 treatment increased CFR from 9.6 ± 9.3 (IgG) to 84.0 ± 23% in the left ventricle in intact, aged animals. These findings suggest that the influence of Thbs-1 and CD47 on coronary perfusion increases with aging and may be therapeutically targeted to reverse coronary microvascular dysfunction.

Adaptation of the Coronary Microcirculation in Aging.

Advancing age will affect every individual and its impact on health deserves significant attention particularly as we address therapeutic possibilities to pathological conditions. The changes that occur in the coronary vasculature as a result of aging‐related senescence set the stage upon which CVD and ischemia can escalate. Because of its importance in health, the consequences of aging on vasculature adaptation must be considered as we identify molecular targets and cell therapies for older patients. To understand the complex relationships between the coronary vasculature and the myocardium, it is important to characterize the unique aged cardiac environment in both locales independent of overlying disease. Therefore, the overall theme of this review is to highlight the biology of aging coronary vasculature and how this promotes a decreased plasticity, exacerbating insults such as ischemia. We will identify potential age‐related mechanisms that may contribute to this overall loss of adaptation and regeneration and review potential therapeutic strategies to ameliorate this dysfunction.

Adipose-Derived Stromal Vascular Fraction Cell Effects on a Rodent Model of Thin Endometrium.

Endometrial dysfunction affects approximately 1% of infertile women, and there is currently no standard therapy for improving fertility treatment outcomes in these patients. In our study, we utilized a rodent model of thin endometrium to test whether intrauterine application of adipose-derived stromal vascular fraction cells (SVF) could improve morphological and physiological markers of endometrial receptivity. Using anhydrous ethanol, endometrial area and gland density were significantly reduced in our model of thin endometrium. Application of SVF was associated with a 29% reduction in endometrial vascular endothelial growth factor (VEGF) expression and significant increases in uterine artery systolic/diastolic velocity ratios and resistance index values, suggesting reduced diastolic microvascular tone. However, no significant improvements in endometrial area or gland density were observed following SVF treatment. 3D confocal imaging demonstrated poor engraftment of SVF cells into recipient tissue, which likely contributed to the negative results of this study. We suspect modified treatment protocols utilizing adjuvant estrogen and/or tail vein cell delivery may improve SVF retention and therapeutic response in subsequent studies. SVF is an easily-obtainable cell product with regenerative capability that may have a future role in the treatment of infertile women with endometrial dysfunction.

Loss of NHERF-1 Expression Prevents Dopamine-Mediated Na-K-ATPase Regulation in Renal Proximal Tubule Cells from Rat Models of Hypertension: Aged F344 Rats and Spontaneously Hypertensive Rats.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

Targeting the Vessel Underdogs: Therapeutic Approaches for Microvessel Dysfunction in the Heart

From an obscure and overlooked beginning, the function of the microvessels in the heart has received increasing attention after the Womens Ischemia Syndrome Evaluation study concluded roughly a decade ago. This review defines the contribution of the coronary microcirculation in the development of heart disease and focuses on the therapeutic methods to reverse coronary microvascular dysfunction. Tissue engineering approaches in the past have largely neglected vascular cells in the attempts to design augmented myocardial tissue, but groups are now making advances that incorporate a functional microcirculation with cardiomyocytes that may advance this line of research. This review covers the definition and classification of coronary microvascular disease, as well as the successful (and unsuccessful) therapeutic approaches in the literature.