Masudul Haque

Involvement of Tumor Necrosis Factor-α in Angiotensin II–Mediated Effects on Salt Appetite, Hypertension, and Cardiac Hypertrophy

Hypertension is considered a low-grade inflammatory condition induced by various proinflammatory cytokines, including tumor necrosis factor (TNF)-α. Recent studies have implicated an involvement of TNF-α in the development of salt-sensitive hypertension induced by angiotensin II (Ang II). To understand further the relationship between TNF-α and Ang II, we examined the responses to Ang II in TNF-α knockout (TNF-α−/−) mice in the present study. A continuous infusion of Ang II (1 μg/kg per minute) for 2 weeks was given to both TNF-α−/− and wild-type (WT) mice with implanted osmotic minipumps. Daily measurement of water intake, salt intake, and urine output were performed using metabolic cages. Blood pressure was monitored continuously with implanted radiotelemetry. Ang II administration for 2 weeks caused increases in salt (0.2±0.07 to 5.6±0.95 mL/d) and water (5.4±0.34 to 11.5±1.2 mL/d) intake and in mean arterial pressure (115±1 to 151±3 mm Hg) in wild-type mice, but these responses were absent in TNF-α−/− mice (0.2±0.04 to 0.3±0.09 mL/d, 5.5±0.2 to 6.1±0.07 mL/d, and 113±2 to 123±3 mm Hg, respectively). Cardiac hypertrophy induced by Ang II was significantly attenuated in TNF-α−/− mice compared with wild-type mice. In a group of TNF-α−/− mice, when replacement therapy was made with recombinant TNF-α, Ang II induced similar responses in salt appetite, mean arterial pressure, and cardiac hypertrophy, as observed in wild-type mice. These results suggest that TNF-α plays a mechanistic role in mediating chronic Ang II–induced effects on salt appetite and blood pressure, as well as on cardiac hypertrophy.

Chronic NF-κB blockade reduces cytosolic and mitochondrial oxidative stress and attenuates renal injury and hypertension in SHR

Nuclear factor-kappaB (NF-kappaB) plays an important role in hypertensive renal injury; however, its roles in perpetuating mitochondrial oxidative stress and renal dysfunction remain unclear. In this study, we assessed the effects of chronic NF-kappaB blockade with pyrrolidine dithiocarbamate (PDTC) on renal dysfunction and mitochondrial redox status in spontaneously hypertensive rats (SHR). PDTC (150 mg.kg body wt(-1).day(-1)) or vehicle was administered orally to 8-wk-old SHR and their respective controls for 15 wk. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography at the start of and at every third week throughout the study. After 15 wk of treatment, anesthetized rats underwent acute renal experiments to determine renal blood flow and glomerular filtration rate using PAH and inulin clearance techniques, respectively. Following renal experiments, kidneys were excised from killed rats, and cortical mitochondria were isolated for reactive oxygen species (ROS) measurements using electron paramagnetic resonance. Tissue mRNA and protein levels of NF-kappaB and oxidative stress genes were determined using real-time PCR and immunofluorescence or Western blotting, respectively. PDTC treatment partially attenuated the increase in SBP (196.4 +/- 9.76 vs. 151.4 +/- 2.12; P < 0.05) and normalized renal hemodynamic and excretory parameters and ATP production rates in SHR. PDTC treatment also attenuated the higher levels of cytosolic and mitochondrial ROS generation and tissue mRNA and protein expression levels of NF-kappaB and oxidative stress genes in SHR without any comparable responses in control rats. These findings suggest that NF-kappaB activation by ROS induces the cytosolic and mitochondrial oxidative stress and tissue injury that contribute to renal dysfunction observed in SHR.

Cytokine blockade attenuates sympathoexcitation in heart failure: cross-talk between nNOS, AT-1R and cytokines in the hypothalamic paraventricular nucleus.

To investigate evidence for the interplay between cytokines, angiotensin II and nNOS in the paraventricular nucleus (PVN), for regulating sympathetic outflow in a rat model of CHF.

Role of Proinflammatory Cytokines and Redox Homeostasis in Exercise-Induced Delayed Progression of Hypertension in Spontaneously Hypertensive Rats

Hypertension is a well-known risk factor for various cardiovascular diseases. Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients. However, the precise mechanisms of exercise training (ExT)–induced effects on the development of hypertension are poorly understood. Therefore, we hypothesized that chronic ExT would delay the progression of hypertension in young spontaneously hypertensive rats (SHRs). In addition, we explored whether the beneficial effects of chronic ExT were mediated by reduced proinflammatory cytokines and improved redox status. We also investigated the involvement of nuclear factor-&kgr;B in exercise-induced effects. To test our hypotheses, young normotensive (Wistar-Kyoto) and SHRs were given moderate-intensity ExT for 16 weeks. Blood pressure was determined by the tail-cuff method, and cardiac function was assessed by echocardiography. Myocardial total reactive oxygen species and superoxide production were measured by electron paramagnetic resonance spectroscopy; tumor necrosis factor-&agr;, interleukin-1&bgr;, gp91phox, and inducible NO synthase by real-time PCR; and nuclear factor &kgr;B activity by electrophoretic mobility shift assay. Chronic ExT in hypertensive rats resulted in significantly reduced blood pressure, reduced concentric hypertrophy, and improved diastolic function. ExT significantly reduced proinflammatory cytokines and inducible NO synthase, attenuated total reactive oxygen species and superoxide production, and increased antioxidants in SHRs. ExT also resulted in increased NO production and decreased nuclear factor &kgr;B activity in SHRs. In summary, chronic ExT delays the progression of hypertension and improves cardiac function in young SHRs; these ExT-induced beneficial effects are mediated by reduced proinflammatory cytokines and improved redox homeostasis via downregulation of nuclear factor-&kgr;B.

Diet-induced renal changes in Zucker rats are ameliorated by the superoxide dismutase mimetic TEMPOL.

Diabetic nephropathy is the leading cause of renal failure in the United States. The obese Zucker rat (OZR; fa/fa) is a commonly used model of type 2 diabetes and metabolic syndrome (MetS), and of the nephropathy and renal oxidative stress commonly seen in these disorders. Heterozygous lean Zucker rats (LZRs; fa/+) are susceptible to high‐fat diet (HFD)‐induced obesity and MetS. The present study was designed to investigate whether 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐N‐oxyl (TEMPOL), a membrane‐permeable radical scavenger, could alleviate the renal effects of MetS in OZR and LZR fed a HFD, which resembles the typical “Western” diet. OZR and LZR were fed a HFD (OZR‐HFD and LZR‐HFD) or regular diet (OZR‐RD and LZR‐RD) and allowed free access to drinking water or water containing 1 mmol/l TEMPOL for 10 weeks. When compared to OZR‐RD animals, OZR‐HFD animals exhibited significantly higher levels of total renal cortical reactive oxygen species (ROS) production, plasma lipids, insulin, C‐reactive protein, blood urea nitrogen (BUN), creatinine (Cr), and urinary albumin excretion (P < 0.05); these changes were accompanied by a significant decrease in plasma high‐density lipoprotein levels (P < 0.05). The mRNA expression levels of desmin, tumor necrosis factor‐α (TNF‐α), nuclear factor κB (NFκB), and NAD(P)H oxidase‐1 (NOX‐1) were significantly higher in the renal cortical tissues of OZR‐HFD animals; NFκB p65 DNA binding activity as determined by electrophoretic mobility shift assay was also significantly higher in these animals. The same trends were noted in LZR‐HFD animals. Our data demonstrate that TEMPOL may prove beneficial in treating the early stages of the nephropathy often associated with MetS.

A major ozonation product of cholesterol, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al, induces apoptosis in H9c2 cardiomyoblasts

Cholesterol, a major neutral lipid component of biological membranes and the lung epithelial lining fluids, is susceptible to oxidation by reactive oxygen and nitrogen species including ozone. The oxidation by ozone in biological environments results in the formation of 3β‐hydroxy‐5‐oxo‐5,6‐secocholestan‐6‐al (cholesterol secoaldehyde or CSeco, major product) along with some other minor products. Recently, CSeco has been implicated in the pathogenesis of atherosclerosis and Alzheimers disease. In this communication, we report that CSeco induces cytotoxicity in H9c2 cardiomyoblasts with an IC50 of 8.9 ± 1.29 μM (n = 6). The observed effect of CSeco at low micromolar concentrations retained several key features of apoptosis, such as changes in nuclear morphology, phosphatidylserine externalization, DNA fragmentation, and caspase 3/7 activity. Treatment of cardiomyocytes with 5 μM CSeco for 24 h, for instance, resulted in 30.8 ± 3.28% apoptotic and 1.8 ± 1.11% of necrotic cells as against DMSO controls that only showed 1.3 ± 0.33% of apoptosis and 1.6 ± 0.67% of necrosis. In general, the loss of cellular viability paralleled the increased occurrence of apoptotic cells in various CSeco treatments. This study, for the first time, demonstrates the induction of apoptotic cell death in cardiomyocytes by a cholesterol ozonation product, implying a role for ozone in myocardial injury.

Interaction of TNF with Angiotensin II Contributes to Mitochondrial Oxidative Stress and Cardiac Damage in Rats

Recent evidence suggests that tumor necrosis factor alpha (TNF) and angiotensin II (ANGII) induce oxidative stress contribute to cardiovascular disease progression. Here, we examined whether an interaction between TNF and ANGII contributes to altered cardiac mitochondrial biogenesis and ATP production to cause cardiac damage in rats. Rats received intraperitoneal injections of TNF (30 µg/kg), TNF + losartan (LOS, 1 mg/kg), or vehicle for 5 days. Left ventricular (LV) function was measured using echocardiography. Rats were sacrificed and LV tissues removed for gene expression, electron paramagnetic resonance and mitochondrial assays. TNF administration significantly increased expression of the NADPH oxidase subunit, gp91phox, and the angiotensin type 1 receptor (AT-1R) and decreased eNOS in the LV of rats. Rats that received TNF only had increased production rates of superoxide, peroxynitrite and total reactive oxygen species (ROS) in the cytosol and increased production rates of superoxide and hydrogen peroxide in mitochondria. Decreased activities of mitochondrial complexes I, II, and III and mitochondrial genes were observed in rats given TNF. In addition, TNF administration also resulted in a decrease in fractional shortening and an increase in Tei index, suggesting diastolic dysfunction. TNF administration with concomitant LOS treatment attenuated mitochondrial damage, restored cardiac function, and decreased expression of AT1-R and NADPH oxidase subunits. Mitochondrial biogenesis and function is severely impaired by TNF as evidenced by downregulation of mitochondrial genes and increased free radical production, and may contribute to cardiac damage. These defects are independent of the downregulation of mitochondrial gene expression, suggesting novel mechanisms for mitochondrial dysfunction in rats given TNF.

Comparison of infrapatellar and subcutaneous adipose tissue stromal vascular fraction and stromal/stem cells in osteoarthritic subjects

Since inflammatory mechanisms have been postulated to link obesity to osteoarthritis, the current study evaluated the ratio of immune cells to multipotent stromal cells within the infrapatellar fat pad (IPFP) and subcutaneous adipose tissue (SQ) of the knee; each depot has potential as a source of regenerative cells. The immunophenotypes of stromal vascular fraction (SVF) and adipose‐derived stem cells (ASCs) of the IPFP and SQ were determined in tissues from osteoarthritic subjects (n = 7) undergoing total knee replacement. Based on a subset of surface antigens, the immunophenotype of ASCs from SQ of OA subjects was not significantly different from that of relatively healthy and leaner subjects undergoing elective liposuction surgery. Flow‐cytometry comparison of SVF cell populations in the IPFP of OA subjects resembled those within the subjects own matched SQ, with the exception of the endothelial marker CD31+, which was significantly greater in cells from SQ. In the OA subjects, lower numbers of capillary‐like structures and higher numbers of stromal and alkaline phosphatase colony‐forming units in the IPFP vs SQ were consistent with this finding; however, ASCs from both depots in OA subjects exhibited comparable adipogenic and osteogenic differentiation potential. Thus, the IPFP contains an ASC and immune cell population similar to that of donor‐matched SQ, making it an alternative ASC source for tissue regeneration. Further studies will be needed to determine whether IPFP immune cell infiltrates play an aetiological role in osteoarthritis equivalent to that shown in diabetes associated with obesity. Copyright

Localisation of 11β‐Hydroxysteroid Dehydrogenase Type 2 in Mineralocorticoid Receptor Expressing Magnocellular Neurosecretory Neurones of the Rat Supraoptic and Paraventricular Nuclei

An accumulating body of evidence suggests that the activity of the mineralocorticoid, aldosterone, in the brain via the mineralocorticoid receptor (MR) plays an important role in the regulation of blood pressure. MR was recently found in vasopressin and oxytocin synthesising magnocellular neurosecretory cells (MNCs) in both the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus. Considering the physiological effects of these hormones, MR in these neurones may be an important site mediating the action of aldosterone in blood pressure regulation within the brain. However, aldosterone activation of MR in the hypothalamus remains controversial as a result of the high binding affinity of glucocorticoids to MR at substantially higher concentrations compared to aldosterone. In aldosterone‐sensitive epithelia, the enzyme 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2) prevents glucocorticoids from binding to MR by converting glucocorticoids into inactive metabolites. The present study aimed to determine whether 11β‐HSD2, which increases aldosterone selectivity, is expressed in MNCs. Specific 11β‐HSD2 immunoreactivity was found in the cytoplasm of the MNCs in both the SON and PVN. In addition, double‐fluorescence confocal microscopy demonstrated that MR‐immunoreactivity and 11β‐HSD2‐in situ hybridised products are colocalised in MNCs. Lastly, single‐cell reverse transcriptase‐polymerase chain reaction detected MR and 11β‐HSD2 mRNAs from cDNA libraries derived from single identified MNCs. These findings strongly suggest that MNCs in the SON and PVN are aldosterone‐sensitive neurones.

Effect of dietary salt intake on epithelial Na+ channels (ENaC) in vasopressin magnocellular neurosecretory neurons in the rat supraoptic nucleus

A growing body of evidence suggests that epithelial Na+ channels (ENaCs) in the brain play a significant role in the regulation of blood pressure; however, the brain structures that mediate the effect are not well understood. Because vasopressin (VP) neurons play a pivotal role in coordinating neuroendocrine and autonomic responses to maintain cardiovascular homeostasis, a basic understanding of the regulation and activity of ENaC in VP neurons is of great interest. We show that high dietary salt intake caused an increase in the expression and activity of ENaC which resulted in the steady state depolarization of VP neurons. The results help us understand one of the mechanisms underlying how dietary salt intake affects the activity of VP neurons via ENaC activity.