Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.
Inspired by studies demonstrating the potential for new myocyte formation within adult mammalian hearts, an ongoing explosion of research is elucidating the biology of cardiac myogenesis and angiogenesis. Multiple lines of research suggest that disease-associated activation of endogenous cardiac repair processes are often insufficient to overcome the cell death resulting from myocardial infarction and chronic heart failure. In this context, this review highlights current evidence supporting endogenous cardiac repair mechanisms in human hearts, recent progress with clinical application of myocardial cell therapy, and complementary efforts to manipulate endogenous myocardial repair processes using a variety of tissue engineering strategies. The goal of this overview is to demonstrate that the insights and opportunities derived from each of these lines of inquiry are mutually complementary for ultimately achieving the goal of therapeutic cardiac regeneration.
Rationale-Clinical trials infusing Bone Marrow Cells (BMCs) into injured hearts have produced measureable improvements in cardiac performance, but were insufficient to improve patient outcomes. Low engraftment rates are cited as probable contributor to limited improvements.Objective-To understand the mechanisms that control myocardial engraftment of BMCs following ischemia-reperfusion injury.Methods and Results-In isolated-perfused mouse hearts, stop-flow ischemia was followed by variable-duration reperfusion (0-60 min) before addition of labeled syngenic BMCs to the perfusate. After a buffer-only wash, the heart was disaggregated. Retained BMCs (digest) and infused BMCs (aliquot) were compared by flow cytometry for c-kit and CD45 expression to determine the proportion of cell subtypes engrafted versus delivered (selectivity ratio). In these studies, a time-dependent selective retention of c-kit+ cells was apparent starting at 30 minutes of reperfusion, at which time c-kit+/CD45+ BMCs showed a selectivity ratio of 18±2 (versus 2±1 in sham-ischemic controls). To study the underlying mechanism for this selective retention, neutralizing antibodies for P-selectin or L-selectin were infused into the heart preparation and incubated with BMCs prior to BMC infusion. Blocking P-selectin in ischemic hearts ablated selectivity for c-kit+/CD45+ BMCs at 30 min reperfusion (selectivity ratio of 3±1) while selectivity persisted in the presence of L-selectin neutralization (selectivity ratio of 17±2). To corroborate this finding, a parallel plate flow chamber was used to study capture and rolling dynamics of purified c-kit+ versus c-kit− BMCs on various selectin molecules. C-kit+ BMCs interacted weakly with L-selectin substrates (0.03±0.01% adhered) but adhered strongly to Pselectin (0.28±0.04% adhered). C-kit− BMCs showed intermediate binding regardless of substrate (0.18±0.04% adhered on L-selectin versus 0.17±0.04% adhered on P-selectin).Conclusions-Myocardial ischemia-reperfusion stress induces selective engraftment of c-kit+ bone marrow progenitor cells via P-selectin activation.
RationaleThe capacity for cardiomyocyte regeneration in the healthy adult human heart is fundamentally relevant for both myocardial homeostasis and cardiomyopathy therapeutics. However, estimates of cardiomyocyte turnover rates conflict greatly, with a study employing C14 pulse-chase methodology concluding 1% annual turnover in youth declining to 0.5% with aging and another using cell population dynamics indicating substantial, age-increasing turnover (4% increasing to 20%).ObjectiveCreate a hybrid mathematical model to critically examine rates of cardiomyocyte turnover derived from alternative methodologies.Methods and ResultsExamined in isolation, the cell population analysis exhibited severe sensitivity to a stem cell expansion exponent (20% variation causing 2-fold turnover change) and apoptosis rate. Similarly, the pulse-chase model was acutely sensitive to assumptions of instantaneous incorporation of atmospheric C14 into the body (4-fold impact on turnover in young subjects) while numerical restrictions precluded otherwise viable solutions. Incorporating considerations of primary variable sensitivity and controversial model assumptions, an unbiased numerical solver identified a scenario of significant, age-increasing turnover (4–6% increasing to 15–22% with age) that was compatible with data from both studies, provided that successive generations of cardiomyocytes experienced higher attrition rates than predecessors.ConclusionsAssignment of histologically-observed stem/progenitor cells into discrete regenerative phenotypes in the cell population model strongly influenced turnover dynamics without being directly testable. Alternatively, C14 trafficking assumptions and restrictive models in the pulse-chase model artificially eliminated high-turnover solutions. Nevertheless, discrepancies among recent cell turnover estimates can be explained and reconciled. The hybrid mathematical model provided herein permits further examination of these and forthcoming datasets.
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