Transcriptomic Analysis of Right Ventricular Remodeling in Two Rat Models of Pulmonary Hypertension: Identification and Validation of Epithelial-to-Mesenchymal Transition in Human Right Ventricular Failure.

BACKGROUND: Right ventricular (RV) dysfunction is a significant prognostic determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Despite the importance of RV function in PAH, the underlying molecular mechanisms of RV dysfunction secondary to PAH remain unclear. We aim to identify and compare molecular determinants of RV failure using RNA sequencing of RV tissue from 2 clinically relevant animal models of PAH. METHODS: We performed RNA sequencing on RV from rats treated with monocrotaline or Sugen with hypoxia/normoxia. PAH and RV failure were confirmed by catheterization and echocardiography. We validated the RV transcriptome results using quantitative real-time polymerase chain reaction, immunofluorescence, and Western blot. Immunohistochemistry and immunofluorescence were performed on human RV tissue from control (n=3) and PAH-induced RV failure patients (n=5). RESULTS: We identified similar transcriptomic profiles of RV from monocrotaline- and Sugen with hypoxia-induced RV failure. Pathway analysis showed genes enriched in epithelial-to-mesenchymal transition, inflammation, and metabolism. Histological staining of human RV tissue from patients with RV failure secondary to PAH revealed significant RV fibrosis and endothelial-to-mesenchymal transition, as well as elevated cellular communication network factor 2 (top gene implicated in epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition) expression in perivascular areas compared with normal RV. CONCLUSIONS: Transcriptomic signature of RV failure in monocrotaline and Sugen with hypoxia models showed similar gene expressions and biological pathways. We provide translational relevance of this transcriptomic signature using RV from patients with PAH to demonstrate evidence of epithelial-to-mesenchymal transition/endothelial-to-mesenchymal transition and protein expression of cellular communication network factor 2 (CTGF [connective tissue growth factor]). Targeting specific molecular mechanisms responsible for RV failure in monocrotaline and Sugen with hypoxia models may identify novel therapeutic strategies for PAH-associated RV failure.

The Role of Estrogen Receptors in Cardiovascular Disease.

Cardiovascular Diseases (CVDs) are the leading cause of death globally. More than 17 million people die worldwide from CVD per year. There is considerable evidence suggesting that estrogen modulates cardiovascular physiology and function in both health and disease, and that it could potentially serve as a cardioprotective agent. The effects of estrogen on cardiovascular function are mediated by nuclear and membrane estrogen receptors (ERs), including estrogen receptor alpha (ERα), estrogen receptor beta (ERß), and G-protein-coupled ER (GPR30 or GPER). Receptor binding in turn confers pleiotropic effects through both genomic and non-genomic signaling to maintain cardiovascular homeostasis. Each ER has been implicated in multiple pre-clinical cardiovascular disease models. This review will discuss current reports on the underlying molecular mechanisms of the ERs in regulating vascular pathology, with a special emphasis on hypertension, pulmonary hypertension, and atherosclerosis, as well as in regulating cardiac pathology, with a particular emphasis on ischemia/reperfusion injury, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction.

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail.

Specific phase relation of serotonin and dopamine modulate the hypothalamo-hypophyseal-gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)-Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3ß-hydroxysteroid dehydrogenase [3ß-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.

Neuroendocrine mechanism of food intake and energy regulation in Japanese quail under differential simulated photoperiodic conditions: Involvement of hypothalamic neuropeptides, AMPK, insulin and adiponectin receptors.

Neuroendocrine coordination between the reproductive and energy regulatory hypothalamic circuitries not only tightly regulates food intake and energy expenditure but also maintains the body weight and reproduction. The effect of different simulated photoperiodic conditions on food intake and neuroendocrine mechanism of energy homeostasis in Japanese quail is not investigated till date. Hence, our present study is designed to elucidate the effect of different simulated photoperiodic conditions on food consumption and neuroendocrine mechanism(s) of energy regulation in this poultry species. The alterations in hypothalamic energy balancing neuropeptides (NPY/AgRP/CART), polypeptide hormone precursor (POMC), protein kinase (AMPK-p-AMPK) as well as receptors of insulin and adiponectin [Insulin Receptor (IR), Adiponectin Receptor 1 & 2] have been investigated in photosensitive (PS), scotorefractory (SR),photorefractory (PR) and scotosensitive (SS) quail. Immunofluorescence and western blotting were used to quantify the expression of these peptides and proteins. Results showed increased food consumption and body weight gain, along with increased expression of NPY, AgRP, IR, adiponectin receptors and p-AMPK, decreased CART and POMC in the hypothalamus of photosensitive and scotorefractory quail. While, opposite findings were observed in photorefractory and scotosensitive quail. Hence, this study may suggest the hypothalamic energy channelization towards reproductive axis in photosensitive and scotorefractory quail to support the full breeding conditions, while hypothalamic energy deprivation in photorefractory and scotosensitive quail leads to reproductive quiescence.

Pueraria tuberosa extract inhibits iNOS and IL-6 through suppression of PKC-α and NF-kB pathway in diabetes-induced nephropathy.

OBJECTIVES: Inflammation plays an important role in the pathogenesis of diabetic nephropathy (DN). The aim of this study was to explore the anti-inflammatory role of PTY-2r (extracted from Pueraria tuberosa), on streptozotocin (STZ)-induced DN rats. METHODS: Diabetes was induced by intraperitoneal injection of STZ (55mg/kg) in rats. After 60 days, the rats were randomly divided into three groups (n = 6/each group), namely DN control group 2, DN rats treated with PTY-2r at dose of 100 mg/100 g, group 3 and 50 mg/100 g, group 4, p.o for 20 days. The normal rats were chosen as a normal control (NC) group 1. KEY FINDINGS: In DN rats, the expression of iNOS and inflammatory cytokines (IL-6 and TNF-α) was significantly increased. Raised expression of PKC-α was also found. As NF-kB is the main transcription factor for the inflammatory response-mediated progression of DN, variation in NF-kB expression and its activated phosphorylated derivative (pNF-kB) were also evaluated and increase in expression was obtained in the kidney of DN rats. PTY-2r treatment significantly reversed these changes in dose-dependent manner. CONCLUSIONS: This study suggested that the nephroprotective effect of PTY-2r is possibly due to downregulation of PKC-α and NF-kB pathway and normalizing the expression of inflammatory cytokines and iNOS in the kidney of DN rats.

Age dependent variations in the deep brain photoreceptors (DBPs), GnRH-GnIH system and testicular steroidogenesis in Japanese quail, Coturnix coturnix japonica.

The complex physiology of aging involves a number of molecular and biochemical events, manifested as signs of senescence. Japanese quail is a very unique and advantageous model to study the signs and symptoms of senescence in the central and peripheral modules of HPG axis. In the present study, we have investigated the age dependent variations in hypothalamic deep brain photoreceptors (DBPs), central GnRH-I/II-GnIH-Mel1cR system, testicular GnRH-GnIH system, testicular steroidogenic genes and proteins, androgen receptor (AR) and serum testosterone level in quail of different age groups [3-wk (sexually immature), 6-wk (sexually mature and crossed the puberty), 16-wk (adult, sexually active and showing full breeding phase) and 144-wk (aged)]. Findings of our present study showed the differential expression of these genes/proteins in quail of different age groups. The low levels of the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain, significantly decreased testicular GnRH/GnRH-R-GnIH, steroidogenic genes/proteins and serum testosterone were observed in immature quail. The significantly increased expression of opsins in the DBPs, GnRH-I, GnIH, Mel1cR in hypothalamus and GnRH-II in midbrain influences the testicular GnRH-GnIH and stimulate the testicular steroidogenesis in mature and adult quail. In aged quail, the significantly decreased levels of hypothalamic DBPs, GnRH-I, GnIH, Mel1cR and midbrain GnRH-II modulates the testicular GnRH-GnIH and further suppresses the genes/proteins involved in steroidogenesis and results in reduced serum testosterone. Hence, it can be concluded from our findings that the testicular steroidogenesis and its neuroendocrine regulation varies with age, in Japanese quail.

From the Cover: 2.45-GHz Microwave Radiation Impairs Hippocampal Learning and Spatial Memory: Involvement of Local Stress Mechanism-Induced Suppression of iGluR/ERK/CREB Signaling.

Microwave (MW) radiation induced oxidative stress reduces dendritic arborization, spine density and number of hippocampal pyramidal neurons and hence, impair learning and spatial memory through p53-dependent/independent apoptosis of hippocampal neuronal and nonneuronal cells. However, the mechanisms responsible for MW radiation induced impairment in memory formation remains still unknown. This study elucidates the effect of short (15 days) and long-term (30 and 60 days) low level 2.45 GHz MW radiation-induced local stress on the hippocampal spatial memory formation pathway in adult male mice. Twelve-weeks old mice were exposed to 2.45 GHz MW radiation (continuous-wave with overall average Power density of 0.0248 mW/cm2 and overall average whole body SAR value of 0.0146 W/Kg) @ 2 h/d for 15, 30, and 60 days. Learning and spatial memory was assessed by 8-arm radial maze. We have investigated the alterations in serum corticosterone level and the expression of glucocorticoid receptor, corticotropin-releasing hormone (CRH), inducible nitric oxide synthase (i-NOS), iGluRs, PSD-95-neuronal NOS (n-NOS) system, protein kinase A, protein kinase Cε-ERK1/2-pERK1/2 in all the hippocampal subregions, viz. CA1, CA2, CA3, and DG through immunohistochemistry/immunofluorescence and alterations in the expression of hippocampal glucocorticoid receptor, CRH-receptor 1 (CRH-R1), cAMP-response element-binding (CREB), and phosphorylated-CREB (p-CREB) through western blot analysis. We observed that 2.45 GHz MW irradiated mice showed slow learning and significantly increased number of working and reference memory errors in radial maze task. Further, 2.45 GHz MW radiation exposure increases serum corticosterone level and the expression of CRH, CRH-R1, and i-NOS, while the expression of iGluRs, n-NOS, PSD-95, protein kinase Cε, protein kinase A, ERK-p-ERK, CREB, and p-CREB decreases in above mentioned hippocampal subregions in a duration dependent manner. Our findings led us to conclude that 2.45 GHz MW radiation exposure induced local stress suppresses signaling mechanism(s) of hippocampal memory formation.

Simulated photoperiod influences testicular activity in quail via modulating local GnRHR-GnIHR, GH-R, Cnx-43 and 14-3-3.

The hypothalamo-hypophyseal-gonadal axis mediated differential photosexual responses in quail kept under different simulated photoperiodic conditions have been studied in details. Local testicular GnRH-GnIH and their receptor system has been hypothesized to be modulated in quail showing different photo-sexual responses and thus influence the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we studied the expression of gonadotropin releasing hormone receptor (GnRH-R), gonadotropin inhibiting hormone receptor (GnIH-R) mRNA, growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), 14-3-3, Connexin-43 (Cnx-43), steroidogenic factor-1 (SF-1), Steroidogenic Acute Regulatory protein (StAR), steroidogenic enzyme (3ß HSD) in testis as well as androgen receptor (AR) in testis and epididymis of photosensitive (PS), scotorefractory (SR), photorefractory (PR) and scotosensitive (SS) quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Connexin-43, SF-1, StAR, 3ß HSD in testis as well as AR in testis and epididymis of PR and SS quail, while PS and SR quail exhibited the opposite results i.e., significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß HSD in testis as well as AR in testis and epididymis. The significantly increased intra-testicular testosterone has been observed in the PS and SR quail while, PR and SS quail showed opposite results. Hence, we conclude that PS and SR quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in PR and SS quail.

Apoptotic mechanism behind the testicular atrophy in photorefractory and scotosensitive quail: Involvement of GnIH induced p-53 dependent Bax-Caspase-3 mediated pathway.

In most of the avian species, daylength or photoperiod is the main environmental factor regulating reproduction. During their annual gonadal cycle, birds once sensitive to short or long day effect develop refractoriness to the same daylength and gonad develop or regress accordingly. The present study investigated the effects of photoperiodic alterations on apoptosis mediated testicular responses of photosensitive/photorefractory and scotosensitive/scotorefractory quail, Coturnix coturnix japonica. Testicular apoptosis in the quail of different photoperiodic conditions was assessed by monitoring the alterations in the local testicular expression of GnRH-I, GnIH, pro-apoptotic proteins (p53 and Bax), inactive caspase (pro-Caspase-3), executioner active-Caspase-3 and inactive/uncleaved PARP-1 (DNA repair enzyme) and TUNEL analysis. Alterations in these parameters indicate that testicular quiescence/regression in scotosensitive and photorefractory quail is mediated by apoptosis of testicular cells and hence apoptosis appears to be the key mechanism of testicular regression in Japanese quail. Present findings demonstrated the underlying molecular mechanism of how avian testes respond differentially to same photoperiodic conditions and exhibit scoto-/photo-sensitivity and refractoriness. It is concluded that photoperiod induced testicular stimulation in photosensitive/scotorefractory quail may be due to apoptotic inhibition and testicular regression in scotosensitive/photorefractory quail is guided by apoptosis, an effect invariably regulated by local action of GnRH and GnIH.

Testicular atrophy and reproductive quiescence in photorefractory and scotosensitive quail: Involvement of hypothalamic deep brain photoreceptors and GnRH-GnIH system.

Birds time their daily and seasonal activities in synchronization with circadian and annual periodicities in the environment, which is mainly provided by changes in photoperiod/day length conditions. Photoperiod appears to act at the level of eye, pineal and encephalic/deep brain photoperception and thus entrain the hypothalamic clock as well as reproductive circuitry in different avian species. In this article our focus of study is to elucidate out the underlying molecular mechanism of modulation of the hypothalamic reproductive circuitry following the photoperception through the hypothalamic photoreceptor cells and the subsequent alteration in the reproductive responses in quail, kept under different simulated photoperiodic conditions. Present study investigated the different simulated photoperiodic conditions induced hypothalamic DBP-GnRH-GnIH system mediated translation of photoperiodic information and subsequent exhibition of differential photosexual responses (scoto-/photo-sensitivity and refractoriness) in Japanese quail, Coturnix coturnix japonica. Paired testes weight and paired testicular volume increased 15.9 and 22.6-fold respectively in scotorefractory quail compare to that of scotosensitive phase and 12.8 and 24.3-fold in photosensitive quail compare to that of photorefractory phase. The pineal/eye melatonin (through melatonin receptor subtype Mel1cR) and hypothalamic deep brain photoreceptor (DBPs) cells directly modulate the hypothalamic GnRH-I/II and GnIH system and thus exhibit testicular stimulation or regression in response to different photoperiodic conditions (PS, PR, SS and SR). The hypothalamic alteration of DBP(s) and GnRH-GnIH system thus may induce the testicular stimulation in PS and SR quail and testicular regression in SS and PR quail.

Effect of extract of Pueraria tuberosa on expression of hypoxia inducible factor-1α and vascular endothelial growth factor in kidney of diabetic rats.

BACKGROUNDS: Kidney hypoxia represents a unifying mechanism in the pathogenesis of diabetic nephropathy. Hypoxia-induced factor (HIF)-1α mediates the metabolic responses of renal hypoxia by modulating the expression of VEGF. In the present study, we investigated the effect of Pueraria tuberosa extract (PTY-2r) on the expression of HIF-1α, VEGF and nephrin in streptozotocin (STZ) induced diabetic nephropathy (DN). METHODS: The model of diabetic nephropathy (DN) was produced by intraperitoneal injection of 55mg/kg of STZ and maintained for 60days. These DN-rats were randomly divided into three groups, i.e., DN, DN+PTY-2r (100mg/100g), and DN+PTY-2r (50mg/100g). A normal control (NC) group was administrated with drug vehicle. Expression of HIF-1α, VEGF and nephrin were evaluated in the renal tissue. RESULTS: Blood glucose, urine protein, serum creatinine, and urea, level were significantly raised along with decreased creatinine clearance in DN rats. Immunofluorescence and Western blot analysis showed significantly increased expression of HIF-1α & VEGF and decreased expression of nephrin in DN control rats. The PTY-2r treatment significantly reversed these changes in a dose-dependent manner. Correlation analysis showed that the expression of VEGF was positively correlated with that of HIF-1α and negatively correlated with nephrin. CONCLUSIONS: The PTY-2r can improve the chronic hyperglycemic condition induced kidney damage, and may delay the development of diabetic nephropathy by inhibiting the expression of HIF-1α and VEGF, thereby restoring the expression of nephrin.

Apoptosis-mediated testicular alteration in Japanese quail (Coturnix coturnix japonica) in response to temporal phase relation of serotonergic and dopaminergic oscillations.

Reproductive performance of many avian species, including Japanese quail, is reported to be modulated by specific temporal phase relation of serotonergic and dopaminergic oscillations. Accordingly, it has been shown that the serotonin precursor 5-HTP and the dopamine precursor l-DOPA given 8 h apart induce gonadal suppression and given 12 h apart lead to gonadal stimulation, while other temporal relationships were found to be ineffective. In the present study, we investigated the effects of 8- and 12-h phase relation of neural oscillations on testicular responses including expression of GnRH-I, GnIH, pro-apoptotic proteins (p53 and Bax), inactive and active executioner caspase-3, and the uncleaved DNA repair enzyme PARP-1. Testicular volume and mass decreased significantly in 8-h quail and increased in 12-h quail compared with controls. Expression of ir-GnIH, p53, Bax and active-caspase-3 increased and that of GnRH-I, pro-caspase-3 and uncleaved PARP-1 decreased in 8-h quail compared with controls. A TUNEL assay also confirmed testicular regression in these quail. Testes of 12-h quail exhibited significantly increased expression of GnRH-I, pro-caspase-3 and uncleaved PARP-1 compared with the control group. Our findings suggest that differential response of avian testes to 8- and 12-h phase relation of serotonergic and dopaminergic neural oscillations may be attributed to autocrine/paracrine action of GnIH expression, which is upregulated in regressed testes, leading to apoptotic changes, and downregulated in developed testes, causing apoptotic inhibition. It is concluded that specific phase relation of neural oscillations may modulate the local testicular GnRH-GnIH system and alter the apoptotic mechanism in quail testes. Moreover, these findings highlight the physiological effects of time-dependent drug delivery, including the specific time intervals between two drugs.

2.45 GHz Microwave Radiation Impairs Learning and Spatial Memory via Oxidative/Nitrosative Stress Induced p53-Dependent/Independent Hippocampal Apoptosis: Molecular Basis and Underlying Mechanism.

A close association between microwave (MW) radiation exposure and neurobehavioral disorders has been postulated but the direct effects of MW radiation on central nervous system still remains contradictory. This study was performed to understand the effect of short (15 days) and long-term (30 and 60 days) low-level MW radiation exposure on hippocampus with special reference to spatial learning and memory and its underlying mechanism in Swiss strain male mice, Mus musculus. Twelve-weeks old mice were exposed to 2.45 GHz MW radiation (continuous-wave [CW] with overall average power density of 0.0248 mW/cm(2) and overall average whole body specific absorption rate value of 0.0146 W/Kg) for 2 h/day over a period of 15, 30, and 60 days). Spatial learning and memory was monitored by Morris Water Maze. We have checked the alterations in hippocampal oxidative/nitrosative stress, neuronal morphology, and expression of pro-apoptotic proteins (p53 and Bax), inactive executioner Caspase- (pro-Caspase-3), and uncleaved Poly (ADP-ribose) polymerase-1 in the hippocampal subfield neuronal and nonneuronal cells (DG, CA1, CA2, and CA3). We observed that, short-term as well as long-term 2.45 GHz MW radiation exposure increases the oxidative/nitrosative stress leading to enhanced apoptosis in hippocampal subfield neuronal and nonneuronal cells. Present findings also suggest that learning and spatial memory deficit which increases with the increased duration of MW exposure (15 < 30 < 60 days) is correlated with a decrease in hippocampal subfield neuronal arborization and dendritic spines. These findings led us to conclude that exposure to CW MW radiation leads to oxidative/nitrosative stress induced p53-dependent/independent activation of hippocampal neuronal and nonneuronal apoptosis associated with spatial memory loss.

Nesfatin-1: Localization and expression in avian gonads and its modulation by temporal phase relation of neural oscillations in female Japanese quail, Coturnix coturnix japonica.

In a search for new appetite-controlling signals, the peptide nesfatin-1, expressed in the brain and peripheral tissues of rodents and humans has been reported to regulate feeding by reducing food intake. Recently it has also been reported that nesfatin-1 might be involved in regulating the reproductive axis in fishes and mammals, but its expression and physiological role if any, is not yet known in birds. In the present study, localization and expression of nesfatin-1 was observed in the testis, ovary and shell gland of poultry species Japanese quail, Coturnix coturnix japonica. Our earlier studies have reported that serotonin precursor 5-HTP and dopamine precursor l-DOPA given 8h apart induces gonadal suppression, when given 12h apart leads to gonadal stimulation while other relationships were found ineffective. In the present study intense ir-nesfatin-1 was observed in the regressed ovary (stromal cells) and shell gland (endometrium) of 8-h Japanese quail while in 12-h quail, weak and scarce immunostaining for nesfatin-1 was detected in the hyperactive ovary and shell gland compared to control. These findings led us to conclude that, an inverse relationship exists between ovarian activity (both in the control and simulated conditions) and nesfatin-1 expression. Present avian study, first of its kind, also suggests the role of nesfatin-1 in reproductive regulation possibly via appetite control and energy balance in female Japanese quail and needs to be investigated further in relation to food intake.