In Vitro Modulation of Spontaneous Activity in Embryonic Cardiomyocytes Cultured on Poly(vinyl alcohol)/Bioglass Type 58S Electrospun Scaffolds.

Because of the physiological and cardiac changes associated with cardiovascular disease, tissue engineering can potentially restore the biological functions of cardiac tissue through the fabrication of scaffolds. In the present study, hybrid nanofiber scaffolds of poly (vinyl alcohol) (PVA) and bioglass type 58S (58SiO2-33CaO-9P2O5, Bg) were fabricated, and their effect on the spontaneous activity of chick embryonic cardiomyocytes in vitro was determined. PVA/Bg nanofibers were produced by electrospinning and stabilized by chemical crosslinking with glutaraldehyde. The electrospun scaffolds were analyzed to determine their chemical structure, morphology, and thermal transitions. The crosslinked scaffolds were more stable to degradation in water. A Bg concentration of 25% in the hybrid scaffolds improved thermal stability and decreased degradation in water after PVA crosslinking. Cardiomyocytes showed increased adhesion and contractility in cells seeded on hybrid scaffolds with higher Bg concentrations. In addition, the effect of Ca2+ ions released from the bioglass on the contraction patterns of cultured cardiomyocytes was investigated. The results suggest that the scaffolds with 25% Bg led to a uniform beating frequency that resulted in synchronous contraction patterns.

Increased angiotensin II AT1 receptor mRNA and binding in spleen and lung of AT2 receptor gene disrupted mice.

To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) receptor mRNA and binding expression in tissues from AT(2) receptor gene disrupted (AT(2)(-/-)) female mice, where AT(2) receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT(2) receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT(1) and AT(2) receptors were not expressed in the same cells. In AT(2)(-/-) mice, we found higher AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT(2) receptor function upregulates AT(1) receptor transcription and expression in a tissue-specific manner and in cells not expressing AT(2) receptors. AT(1) upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT(2)(-/-) phenotype, consistent with enhanced AT(1) receptor activation in a number of tissues.

Estrogen upregulates renal angiotensin II AT(2) receptors.

AT(2) receptors may act in opposition to and in balance with AT(1) receptors, their stimulation having beneficial effects. We found renal AT(2) receptor expression in female mice higher than in male mice. We asked the question of whether such expression might be estrogen dependent. In male, female, ovariectomized, and estrogen-treated ovariectomized mice, we studied renal AT(1) and AT(2) receptors by immunocytochemistry and autoradiography, AT(2) receptor mRNA by RT-PCR, and cAMP, cGMP, and PGE(2) by RIA. AT(1) receptors predominated. AT(2) receptors were present in glomeruli, medullary rays, and inner medulla, and in female kidney capsule. AT(1) and AT(2) receptors colocalized in glomeruli. Female mice expressed fewer glomerular AT(1) receptors. Ovariectomy decreased AT(1) receptors in medullary rays and capsular AT(2) receptors. Estrogen administration normalized AT(1) receptors in medullary rays and increased AT(2) receptors predominantly in capsule and inner medulla, and also in glomeruli, medullary rays, and inner stripe of outer medulla. In medullas of estrogen-treated ovariectomized mice there was higher AT(2) receptor mRNA, decreased cGMP, and increased PGE(2) content. We propose that the protective effects of estrogen may be partially mediated through enhancement of AT(2) receptor stimulation.

Protection against ischemia and improvement of cerebral blood flow in genetically hypertensive rats by chronic pretreatment with an angiotensin II AT1 antagonist.

BACKGROUND AND PURPOSE: Pretreatment with angiotensin II AT(1) receptor antagonists protects against cerebral ischemia. We studied whether modulation of cerebral blood flow (CBF) and morphometric changes in brain arteries participated in this protective mechanism. METHODS: We pretreated adult spontaneously hypertensive rats with equally antihypertensive doses of candesartan (0.1 or 0.3 mg/kg per day), nicardipine (0.1 mg/kg per day), or captopril (3.0 mg/kg per day) for 3 or 28 days via subcutaneous osmotic minipumps followed by permanent left middle cerebral artery (MCA) occlusion distal to the origin of the lenticulostriate arteries. We measured CBF by autoradiography with 4-iodo-[N-methyl-(14)C]antipyrine 3 hours after operation and the areas of infarct and tissue swelling 24 hours after operation. Morphometric changes in the MCA were studied after antihypertensive treatment. RESULTS: Twenty-eight days of candesartan pretreatment decreased the infarct area by 31%; reduced the CBF decrease at the peripheral area of ischemia and the cortical volume of severe ischemic lesion, where CBF was <0.50 mL/g per minute; increased the MCA external diameter by 16%; and reduced the media thickness of the MCA by 23%. Captopril pretreatment for 28 days decreased the infarct area by 25%. Pretreatment with candesartan for 3 days or nicardipine for 28 days was ineffective. CONCLUSIONS: Angiotensin II system inhibition protects against neuronal injury more effectively than calcium channel blockade. Protection after AT(1) receptor blockade is not directly correlated with blood pressure reduction but with normalization of MCA media thickness, leading to increased arterial compliance and reduced CBF decrease during ischemia at the periphery of the lesion.

Increased angiotensin II AT(1) receptor expression in paraventricular nucleus and hypothalamic-pituitary-adrenal axis stimulation in AT(2) receptor gene disrupted mice.

Angiotensin II AT(2) receptor gene-disrupted mice have increased blood pressure and response to angiotensin II, behavioral alterations, greater response to stress, and increased adrenal AT(1) receptors. We studied hypothalamic AT(1) receptor binding and mRNA by receptor autoradiography and in situ hybridization, adrenal catecholamines by HPLC, adrenal tyrosine hydroxylase mRNA by in situ hybridization and pituitary and adrenal hormones by RIA in AT(2) receptor-gene disrupted mice and wild-type controls. To confirm the role of adrenal AT(1) receptors, we treated wild-type C57 BL/6J mice with the AT(1) antagonist candesartan for 2 weeks, and measured adrenal hormones, catecholamines and tyrosine hydroxylase mRNA. In the absence of AT(2) receptor transcription, we found increased AT(1) receptor binding in brain areas involved in the regulation of the hypothalamic-pituitary-adrenal axis, the hypothalamic paraventricular nucleus and the median eminence, and increased adrenal catecholamine synthesis as shown by higher adrenomedullary tyrosine hydroxylase mRNA and higher adrenal dopamine, norepinephrine and epinephrine levels when compared to wild-type mice. In addition, in AT(2) receptor gene-disrupted mice there were higher plasma adrenocorticotropin (ACTH) and corticosterone levels and lower adrenal aldosterone content when compared to wild-type controls. Conversely, AT(1) receptor inhibition in CB57 BL/6J mice reduced adrenal tyrosine hydroxylase mRNA and catecholamine content and increased adrenal aldosterone content. These results can help to explain the enhanced response of AT(2) receptor gene-disrupted mice to exogenous angiotensin II, support the hypothesis of cross-talk between AT(1) and AT(2) receptors, indicate that the activity of the hypothalamic-pituitary-adrenal axis parallels the AT(1) receptor expression, and suggest that expression of AT(1) receptors can be dependent on AT(2) receptor expression. Our results provide an explanation for the increased sensitivity to stress in this model.

Restraint stress modulates brain, pituitary and adrenal expression of angiotensin II AT(1A), AT(1B) and AT(2) receptors.

Angiotensin II (Ang II) AT(1) receptors are involved in the regulation of the stress response. In adult male rats, acute restraint increased AT(1A) mRNA in paraventricular nucleus. Repeated restraint increased AT(1A) mRNA and AT(1) binding in paraventricular nucleus and AT(1) binding in subfornical organ and median eminence. AT(1B) and AT(2) receptors were not expressed in brain areas involved in the stress response. Acute restraint increased anterior pituitary AT(1A) mRNA and AT(1) binding and decreased AT(1B) mRNA. During repeated restraint, the increase in AT(1A) mRNA in the anterior pituitary was maintained, but AT(1B) mRNA and AT(1) binding returned to normal levels. In adrenal zona glomerulosa, AT(1B) mRNA, AT(1) binding, AT(2) mRNA and AT(2) binding decreased during acute restraint. Receptor mRNA and binding returned to normal after repeated stress, with the exception of rebound increase in adrenal zona glomerulosa AT(2) mRNA. In adrenal medulla, AT(1A) mRNA increased and AT(2) mRNA decreased during acute restraint. AT(1A) mRNA remained increased during repeated restraint, while alterations in AT(2) mRNA were no longer present. Expression of AT(1A), AT(1B) and AT(2) receptors in the hypothalamic-pituitary-adrenal axis is tissue specific and is different in acute and repeated stress. Increased brain, pituitary and adrenomedullary AT(1A) receptor expression correlates with hypothalamic-pituitary-adrenal axis stimulation, supporting the hypothesis of Ang II, through selective AT(1A) receptor stimulation, as an important determinant of the acute and repeated stress response. Decreased adrenal zona glomerulosa and anterior pituitary AT(1B) receptors during acute stress can be interpreted as compensatory to increased stimulation by Ang II. There may be additional roles for adrenal AT(2) receptors during acute stress, possibly related to interaction or cross-talk with AT(1) receptors.