Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization.

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

Brain TACE (Tumor Necrosis Factor-α-Converting Enzyme) Contributes to Sympathetic Excitation in Heart Failure Rats.

TNF-α (tumor necrosis factor-α) is initially synthesized as a transmembrane protein that is cleaved by TACE (TNF-α-converting enzyme) to release soluble TNF-α. The elevated level of TNF-α in the brain and circulation in heart failure (HF) suggests an increase in the TACE-mediated ectodomain shedding process. The present study sought to determine whether TACE is upregulated in cardiovascular/autonomic brain regions like subfornical organ and hypothalamic paraventricular nucleus in rats with ischemia-induced HF and whether TACE plays a role in TNF-α-driven sympathetic excitation. We found that TACE was expressed throughout the subfornical organ and paraventricular nucleus, with significantly higher levels in HF than in sham-operated (Sham) rats. Intracerebroventricular injection of recombinant TACE induced a mild increase in blood pressure, heart rate, and renal sympathetic nerve activity that peaked at 15 to 20 minutes in both Sham and HF rats. HF rats had a secondary prolonged increase in these variables that was prevented by the TNF-α inhibitor SPD304. Intracerebroventricular administration of the TACE inhibitor TNF-alpha protease inhibitor 1 decreased blood pressure, heart rate, and renal sympathetic nerve activity in Sham and HF rats, with an exaggerated reduction in heart rate and renal sympathetic nerve activity in the HF rats. Direct microinjection of TACE or TNF-alpha protease inhibitor 1 into paraventricular nucleus or subfornical organ of Sham and HF rats elicited blood pressure, heart rate, and renal sympathetic nerve activity responses similar to intracerebroventricular TACE or TNF-alpha protease inhibitor 1. Intracerebroventricular infusion of Ang II (angiotensin II) and IL (interleukin)-1ß increased TACE expression in subfornical organ and paraventricular nucleus of normal rats. These data suggest that a TACE-mediated increase in soluble TNF-α in the brain contributes to sympathetic excitation in HF.

Sex differences in the central and peripheral manifestations of ischemia-induced heart failure in rats.

Sex differences in the presentation, outcome, and responses to treatment of systolic heart failure (HF) have been reported. In the present study, we examined the effect of sex on central neural mechanisms contributing to neurohumoral excitation and its peripheral manifestations in rats with HF. Male and female Sprague-Dawley rats underwent coronary artery ligation (CL) to induce HF. Age-matched rats served as controls. Ischemic zone and left ventricular function were similar 24 h and 4 wk after CL. Female rats with HF had a lower mortality rate and less hemodynamic compromise, pulmonary congestion, and right ventricular remodeling 4 wk after CL. Plasma angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine levels were increased in HF rats in both sexes, but AVP and norepinephrine levels increased less in female rats. In the hypothalamic paraventricular nucleus, a key cardiovascular-related nucleus contributing to neurohumoral excitation in HF, mRNA levels for the proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß as well as cyclooxygenase-2 and the ANG II type 1a receptor were increased in HF rats of both sexes, but less so in female rats. Angiotensin-converting enzyme 2 protein levels increased in female HF rats but decreased in male HF rats. mRNA levels of AVP were lower in female rats in both control and HF groups compared with the respective male groups. Activation of extracellular signal-regulated protein kinases 1 and 2 increased similarly in both sexes in HF. The results suggest that female HF rats have less central neural excitation and less associated hemodynamic compromise than male HF rats with the same degree of initial ischemic cardiac injury. NEW & NOTEWORTHY Sex differences in the presentation and responses to treatment of heart failure (HF) are widely recognized, but the underlying mechanisms are poorly understood. The present study describes sex differences in the central nervous system mechanisms that drive neurohumoral excitation in ischemia-induced HF. Female rats had a less intense central neurochemical response to HF and experienced less hemodynamic compromise. Sex hormones may contribute to these differences in the central and peripheral adaptations to HF.

Role of myeloperoxidase in abdominal aortic aneurysm formation: mitigation by taurine.

Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.

Central actions of the chemokine stromal cell-derived factor 1 contribute to neurohumoral excitation in heart failure rats.

The ample expression of chemokines and their receptors by neurons in the brain suggests that they play a functional role beyond the coordination of inflammatory and immune responses. Growing evidence implicates brain chemokines in the regulation of neuronal activity and neurohormonal release. This study examined the potential role of brain chemokines in regulating hemodynamic, sympathetic, and neuroendocrine mechanisms in rats with ischemia-induced heart failure (HF). Immunohistochemical analysis revealed that the chemokine stromal cell-derived factor 1 (SDF-1)/CXCL12 was highly expressed in the hypothalamic paraventricular nucleus and subfornical organ and that SDF-1 expression was significantly increased in HF rats compared with sham-operated (SHAM) control rats. ICV injection of SDF-1 induced substantial and long-lasting increases in blood pressure, heart rate, and renal sympathetic nerve activity in both SHAM and HF rats, but responses were exaggerated in HF rats. Bilateral microinjection of SDF-1 into the paraventricular nucleus also elicited exaggerated increases in blood pressure, heart rate, and renal sympathetic nerve activity in the HF rats. A 4-hour ICV infusion of SDF-1 increased plasma levels of arginine vasopressin, adrenocorticotropic hormone, and norepinephrine in normal rats, responses that were prevented by pretreatment with ICV SDF-1 short-hairpin RNA (shRNA). ICV administration of SDF-1 shRNA also reduced plasma arginine vasopressin, adrenocorticotropic hormone, and norepinephrine levels in HF rats. These data suggest that the chemokine SDF-1, acting within the brain, plays an important role in regulating sympathetic drive, neuroendocrine release, and hemodynamic function in normal and pathophysiological conditions and so may contribute to the neural and humoral activation in HF.

Mitogen-activated protein kinases mediate upregulation of hypothalamic angiotensin II type 1 receptors in heart failure rats.

In heart failure (HF), angiotensin II type 1 receptor (AT(1)-R) expression is upregulated in brain regions regulating sympathetic drive, blood pressure, and body fluid homeostasis. However, the mechanism by which brain AT(1)-R are upregulated in HF remains unknown. The present study examined the hypothesis that the angiotensin II (Ang II)-triggered mitogen-activated protein kinases (MAPKs) p44/42, p38, and c-Jun N-terminal kinase contribute to upregulation of the AT(1)-R in the hypothalamus of rats with HF. AT(1)-R protein, AT(1)-R mRNA, and AT(1)-R immunoreactivity increased in the paraventricular nucleus of hypothalamus and the subfornical organ of rats with ischemia-induced HF compared with sham-operated controls. Phosphorylated p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK also increased in paraventricular nucleus and subfornical organ. A 4-week ICV infusion of the AT(1)-R antagonist losartan decreased AT(1)-R protein and phosphorylation of p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK in the HF rats. A 4-week ICV infusion of the p44/42 MAPK inhibitor PD98059 or the c-Jun N-terminal kinase inhibitor SP600125 significantly decreased AT(1)-R protein and AT(1)-R immunoreactivity in the paraventricular nucleus and subfornical organ, but the p38 MAPK inhibitor SB203580 did not. Treatment with ICV losartan, PD98059, and SP600125 had no effect on AT(1)-R expression by Western blot in sham-operated rats. In untreated HF rats 4 weeks after coronary ligation, a 3-hour ICV infusion of PD98059, SP600125, or losartan reduced AT(1)-R mRNA in paraventricular nucleus and subfornical organ. These data indicate that MAPK plays an important role in the upregulation of AT(1)-R in the rat forebrain in HF and suggest that Ang II upregulates its own receptor by this mechanism.

Inhibition of brain proinflammatory cytokine synthesis reduces hypothalamic excitation in rats with ischemia-induced heart failure.

The expression of proinflammatory cytokines increases in the hypothalamus of rats with heart failure (HF). The pathophysiological significance of this observation is unknown. We hypothesized that hypothalamic proinflammatory cytokines upregulate the activity of central neural systems that contribute to increased sympathetic nerve activity in HF, specifically, the brain renin-angiotensin system (RAS) and the hypothalamic-pituitary-adrenal (HPA) axis. Rats with HF induced by coronary ligation and sham-operated controls (SHAM) were treated for 4 wk with a continuous intracerebroventricular infusion of the cytokine synthesis inhibitor pentoxifylline (PTX, 10 microg/h) or artificial cerebrospinal fluid (VEH). In VEH-treated HF rats, compared with VEH-treated SHAM rats, the hypothalamic expression of proinflammatory cytokines was increased, along with key components of the brain RAS (renin, angiotensin-converting enzyme, angiotensin type 1 receptor) and corticotropin-releasing hormone, the central indicator of HPA axis activation, in the paraventricular nucleus (PVN) of the hypothalamus. The expression of other inflammatory/excitatory mediators (superoxide, prostaglandin E(2)) was also increased, along with evidence of chronic neuronal excitation in PVN. VEH-treated HF rats had higher plasma levels of norepinephrine, ANG II, interleukin (IL)-1beta, and adrenocorticotropic hormone, increased left ventricular end-diastolic pressure, and increased wet lung-to-body weight ratio. With the exception of plasma IL-1beta, an indicator of peripheral proinflammatory cytokine activity, all measures of neurohumoral excitation were significantly lower in HF rats treated with intracerebroventricular PTX. These findings suggest that the increase in brain proinflammatory cytokines observed in rats with ischemia-induced HF is functionally significant, contributing to neurohumoral excitation by activating brain RAS and the HPA axis.

Does aldosterone upregulate the brain renin-angiotensin system in rats with heart failure?

The brain renin-angiotensin system (RAS) contributes to increased sympathetic drive in heart failure (HF). The factors upregulating the brain RAS in HF remain unknown. We hypothesized that aldosterone (ALDO), a downstream product of the systemic RAS that crosses the blood-brain barrier, signals the brain to increase RAS activity in HF. We examined the relationship between circulating and brain ALDO in normal intact rats, in adrenalectomized rats receiving subcutaneous infusions of ALDO, and in rats with ischemia-induced HF and sham-operated controls. Brain ALDO levels were proportional to plasma ALDO levels across the spectrum of rats studied. Compared with sham-operated controls rats, HF rats had higher plasma and hypothalamic tissue levels of ALDO. HF rats also had higher expression of mRNA and protein for angiotensin-converting enzyme and angiotensin type 1 receptors in the hypothalamus, increased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide generation in the paraventricular nucleus of the hypothalamus, increased excitation of paraventricular nucleus neurons, and increased plasma norepinephrine. HF rats treated for 4 weeks with intracerebroventricular RU28318 (1 microg/h), a selective mineralocorticoid receptor antagonist, had less hypothalamic angiotensin-converting enzyme and angiotensin type 1 receptor mRNA and protein, less reduced nicotinamide-adenine dinucleotide phosphate-induced superoxide in the paraventricular nucleus, fewer excited paraventricular nucleus neurons, and lower plasma norepinephrine. RU28318 had no effect on plasma ALDO or on angiotensin-converting enzyme or angiotensin type 1 receptor expression in brain cortex. The data demonstrate that ALDO of adrenal origin enters the hypothalamus in direct proportion to plasma levels and suggest that ALDO contributes to the upregulation of hypothalamic RAS activity and sympathetic drive in heart failure.

Novel effect of mineralocorticoid receptor antagonism to reduce proinflammatory cytokines and hypothalamic activation in rats with ischemia-induced heart failure.

Blocking brain mineralocorticoid receptors (MRs) reduces the high circulating levels of tumor necrosis factor (TNF)-alpha in heart failure (HF) rats. TNF-alpha and other proinflammatory cytokines activate neurons in the paraventricular nucleus (PVN) of hypothalamus, including corticotropin-releasing hormone (CRH) neurons, by inducing cyclooxygenase (COX)-2 activity and synthesis of prostaglandin E2 by perivascular cells of the cerebral vasculature. We tested the hypothesis that systemic treatment with a MR antagonist would reduce hypothalamic COX-2 expression and PVN neuronal activation in HF rats. Rats underwent coronary ligation to induce HF, confirmed by echocardiography, or sham surgery, followed by 6 weeks treatment with eplerenone (30 mg/kg per day, orally) or vehicle (drinking water). Eplerenone-treated HF rats had lower plasma TNF-alpha, interleukin (IL)-1beta and IL-6, less COX-2 staining of small blood vessels penetrating PVN, fewer PVN neurons expressing Fra-like activity (indicating chronic neuronal activation), and fewer PVN neurons staining for TNF-alpha, IL-1beta, and CRH than vehicle-treated HF rats. COX-2 and CRH protein expression in hypothalamus were 1.7- and 1.9-fold higher, respectively, in HF+vehicle versus sham+vehicle rats; these increases were attenuated (26% and 25%, respectively) in HF+eplerenone rats. Eplerenone-treated HF rats had less prostaglandin E2 in cerebrospinal fluid, lower plasma norepinephrine levels, lower left ventricular end-diastolic pressure, and lower right ventricle/body weight and lung/body weight ratios, but no improvement in left ventricular function. Treatment of HF rats with anticytokine agents, etanercept or pentoxifylline, produced very similar results. This study reveals a previously unrecognized effect of MR antagonism to minimize cytokine-induced central neural excitation in rats with HF.

Bradycardia induces angiogenesis, increases coronary reserve, and preserves function of the postinfarcted heart.

BACKGROUND: We tested the hypothesis that induction of chronic bradycardia would trigger an upregulation of key growth factors and receptors, which would then lead to angiogenesis and improve coronary reserve in the left ventricle after myocardial infarction. METHODS AND RESULTS: Bradycardia was induced in rats by administering alinidine via osmotic pumps beginning 1 day after coronary artery ligation. Echocardiographic analysis was conducted before and after treatment. Morphometric analysis was used in perfusion-fixed hearts to document arteriolar and capillary growth. Western blots were used to evaluate growth factor and receptor changes. During the first week of treatment, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), and basic fibroblast growth factor proteins were higher in the treated group, whereas VEGF receptor 2 (Flk-1), angiopoietin-1, and angiopoietin-2 were not affected by treatment. After 3 weeks, VEGF protein remained elevated, and bradycardia was associated with a higher capillary length density in the border (40%) and remote (14%) regions and a higher arteriolar length density in the septum (62%), despite a greater increase in left ventricular mass. Although arteriolar length density increased in all size classes, the greatest increase occurred in the smallest (terminal) arterioles. This vascular growth was associated with a 23% greater coronary reserve. Echocardiography revealed a smaller increase in ventricular volume and a greater preservation of ejection fraction in rats treated with bradycardia. CONCLUSIONS: Pharmacologic induction of bradycardia enhances vascularity and coronary reserve, preserves function of surviving myocardium, and therefore, is a noninvasive, therapeutic avenue that provides an alternative to gene therapy.

Heart failure and the brain: new perspectives.

Despite recent therapeutic advances, the prognosis for patients with heart failure remains dismal. Unchecked neurohumoral excitation is a critical element in the progressive clinical deterioration associated with the heart failure syndrome, and its peripheral manifestations have become the principal targets for intervention. The link between peripheral systems activated in heart failure and the central nervous system as a source of neurohumoral drive has therefore come under close scrutiny. In this context, the forebrain and particularly the paraventricular nucleus of the hypothalamus have emerged as sites that sense humoral signals generated peripherally in response to the stresses of heart failure and contribute to the altered volume regulation and augmented sympathetic drive that characterize the heart failure syndrome. This brief review summarizes recent studies from our laboratory supporting the concept that the forebrain plays a critical role in the pathogenesis of ischemia-induced heart failure and suggesting that the forebrain contribution must be considered in designing therapeutic strategies. Forebrain signaling by neuroactive products of the renin-angiotensin system and the immune system are emphasized.