Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors.

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration and improve heart function. However, evidence suggests that their regenerative capacity may be limited in conditions of severe hypoxia. Elucidating the mechanisms involved in CPC protection against hypoxic stress is essential to maximize their cardioprotective and therapeutic potential. We investigated the effects of hypoxic stress on CPCs and found significant reduction in proliferation and impairment of vasculogenesis, which were associated with induction of quiescence, as indicated by accumulation of cells in the G0-phase of the cell cycle and growth recovery when cells were returned to normoxia. Induction of quiescence was associated with a decrease in the expression of c-Myc through mechanisms involving protein degradation and upregulation of p21. Inhibition of c-Myc mimicked the effects of severe hypoxia on CPC proliferation, also triggering quiescence. Surprisingly, these effects did not involve changes in p21 expression, indicating that other hypoxia-activated factors may induce p21 in CPCs. Our results suggest that hypoxic stress compromises CPC function by inducing quiescence in part through downregulation of c-Myc. In addition, we found that c-Myc is required to preserve CPC growth, suggesting that modulation of pathways downstream of it may re-activate CPC regenerative potential under ischemic conditions.

Background: Adult cardiac stem cells (CSCs) capable of self-renewal and differentiation into cardiac, smooth muscle and endothelial lineages have been shown to promote tissue repair after ischemic injury and improve heart function. Despite their potential role in cardiac regeneration, evidence suggests that resident CSCs are impaired in conditions such as myocardial infarction, limiting their regenerative capacity. Methods and Results: The growth rate of CSCs is significantly reduced by 40% (p<0.05) under hypoxic stress (0.5% O 2 ). Using an extracellular-matrix (ECM) and adhesion-focused PCR array, we found that incubation of CSCs for 18 hours under hypoxia leads to down-regulation of several genes, including osteopontin, thrombospondin-1 and integrins. Due to their critical role in communicating extracellular signals regulating stem cell mobilization, proliferation, survival, migration and differentiation we decided to investigate the role of integrins in CSCs self-renewal and differentiation. Our gene expression studies showed that integrin-β1 (Itgb1) is the most abundant integrin expressed in CSCs. Therefore, we knocked down (KD) Itgb1 in CSCs and evaluated these cells regarding adhesion, growth and differentiation properties. Our results showed that reduction in Itgb1 levels in CSCs significantly decreased their interaction with extracellular matrix components fibronectin, laminin and vitronectin by 32%, 20% and 24% (p<0.05), respectively. KD of Itgb1 in CSCs reduced growth by 40% (p<0.05), similar to what we observed by with wild-type cells in hypoxia. Assessment of endothelial differentiation showed that KD cells have a higher angiogenic potential vs. NS-control, as demonstrated by upregulation of vWF (3-fold), VEGF (2.5-fold), Pecam1 (2.3-fold) and Nos3 (1.9-fold) (p<0.05). Interestingly, Itgb1 KD per se induced upregulation of these vascular markers in CSCs. Conclusions: Our results suggest that Itgb1 plays an important role in CSCs self-renewal, differentiation and cell adhesion. It is possible that loss of self-renewal is a consequence of changes in CSCs fate as knockdown of Itgb1 leads to upregulation of endothelial lineage markers in these cells. This would limit the expansion of CSCs and their regenerative capacity.

Introduction: The adult heart harbors a population of resident stem cells capable of self-renewal and differentiation into cardiac, smooth muscle and endothelial lineages. Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration and improve heart function. However, despite their potential use in cardiac repair, evidence suggests that resident CPCs regenerative capacity is limited in conditions of severe hypoxia such as ischemic cardiomyopathy. Elucidation of the mechanisms involved in CPC protection against hypoxic stress is essential to maximize their cardioprotective and therapeutic potential. Methods and Results: We investigated the growth pattern of murine CPCs under normoxia (21% oxygen) and hypoxia (0.5% oxygen) conditions along a period of 96 hours. We found that CPC proliferation in hypoxia was significantly reduced by 40% after 72-96 hours (p<0.05, n=4-7), without evidence of increase in cell death. CPC proliferation was accompanied with a time-dependent increase in senescence-associated β-galactosidase activity by 30% in hypoxia relative to normoxia control (p<0.02, n=7). Western blot analysis of CPCs grown under normoxia and hypoxia revealed a time-dependent decrease in the expression of the transcription factor cMyc and its downstream target Bmi-1 by 40% and 30% (p<0.05, n=4-5) relative to normoxia control, respectively. Interestingly, reduction in cMyc protein expression was not associated with a decrease in cMyc transcripts, suggesting that cMyc protein degradation is activated under hypoxia. Treatment of CPCs with cycloheximide to block protein synthesis showed that the rate of cMyc degradation under hypoxia is increased by 30% (p<0.05, n=5) relative to normoxia. GSK3β plays a central role in the control of cMyc stability. Growth of CPCs in the presence of GSK3β inhibitor rescued proliferation in hypoxia by approximately 40% (n=4). Conclusions: Our results suggest that hypoxic stress reduces CPC self-renewal potential by inducing CPC senescence through a mechanism that involves decrease of cMyc stability through GSK3β activation. Modulation of GSK3β activity may be used as a therapeutic approach for protection of endogenous CPCs self-renewal potential under ischemic conditions.