C-type natriuretic peptide (CNP) inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor growth by modulating inflammation in Swiss albino mice.

C-type natriuretic peptide (CNP) exhibits anti-inflammatory activity besides its natriuretic and diuretic functions. The present study aimed to determine the anticancer and synergistic therapeutic activity of CNP against a 7,12-Dimethylbenz[a]anthracene (DMBA)/Croton oil-induced skin tumor mouse model. CNP (2.5 µg/kg body weight) was injected either alone and/or in combination with Cisplatin (CDDP) (2 mg/kg body weight) for 4 weeks. The dorsal skin tumor incidences/growth and mortality rate were recorded during the experimental period of 16 weeks. The serum C-reactive protein (CRP), and lactate dehydrogenase (LDH) levels, infiltrating mast cells, and AgNORs proliferating cells count were analyzed in control and experimental mice. Further, the expression profile of marker genes of proliferation, inflammation, and progression molecules were analyzed using Reverse transcriptase-polymerase chain reaction (RT-PCR)/quantitative PCR (qPCR), western blot, and immunohistochemistry. The DMBA/Croton oil-induced mice exhibited 100% tumor incidence. Whereas, CNP alone, CDDP alone, and CNP+CDDP combination-treated mice exhibited 58%, 46%, and 24% tumor incidence, respectively. Also, a marked reduction in the levels of serum CRP and LDH, the number of infiltrating mast cells count and AgNORs proliferating cells count were noticed in the mice skin sections. Further, a significant reduction in both mRNA and protein expression levels of proliferation, inflammation, and progression markers were noticed in CNP (p < 0.01), CDDP (p < 0.01), and CNP+CDDP combination (p < 0.001) treated mice, respectively. The results of the present study suggest that CNP has anticancer activity. Further, the CNP+CDDP treatment has more promising anticancer activity as compared with CNP or CDDP alone treatment, probably due to the synergistic antiproliferative and anti-inflammatory activities of CNP and CDDP.

Suppression of Npr1, not Npr2 gene function induces hypertrophic growth in H9c2 cells in vitro.

Npr1 gene (coding for NPR-A) and Npr2 gene (coding for NPR-B) are identified as intrinsic anti-hypertrophic genes that opposes abnormal cardiac remodeling. However, the functional role of Npr1 and Npr2 genes during cardiac hypertrophic growth is not well understood. Hence, the present investigation was aimed to study the effect of Npr1 and Npr2 gene silencing, respectively on ß-AR activation induced cardiac hypertrophic growth in H9c2 cells in vitro. The control, Npr1, and Npr2 gene suppressed H9c2 cells, respectively were treated with ISO (10-5 M) for 48 h. The mRNA and protein expression profile of NPR-A, NPR-B, PKG-I and cGMP were analyzed by qPCR, Western blotting, ELISA, and immunofluorescence methods, respectively. A marked increase in cell size (30.10 ± 0.51 µm vs 61.83 ± 0.43 µm, 2-fold) accompanied by elevated hypertrophic marker genes (α-sk and ß-MHC 3-fold, respectively) expression was observed in Npr1 gene suppressed H9c2 cells as compared with control cells. In contrast, the Npr2 gene suppression in H9c2 cells neither altered the cell size nor the level of hypertrophic marker genes expression. Upon exposure to Isoproterenol, the Npr1 suppressed H9c2 cells exhibited further increase in cell size (1.5 fold), whereas, no significant increase in cell size or marker genes expression was noticed in Npr2 suppressed cells. Moreover, the intracellular cGMP level was down-regulated by 2-fold in Npr1 suppressed cells, while, no significant change was observed in Npr2 suppressed cells. Together, these results suggest that Npr1, not Npr2 gene function is positively associated with the initiation of cardiac fetal gene program and development of cardiac hypertrophic growth.

Angiotensin II down-regulates natriuretic peptide receptor-A expression and guanylyl cyclase activity in H9c2 (2-1) cardiac myoblast cells: Role of ROS and NF-κB.

Atrial natriuretic peptide (ANP)/natriuretic peptide receptor-A (NPR-A) system is suggested as an endogenous anti-hypertrophic protective mechanism of the heart. We have shown previously that Angiotensin II (ANG II), an effector molecule of renin-angiotensin-aldosterone system, down-regulates NPR-A expression and its activity in vivo rat heart. However, the underlying mechanism by which ANG II down-regulates NPR-A expression in the heart is not well understood. Hence, the present investigation was aimed to determine whether ANG II-stimulated reactive oxygen species (ROS) and NF-κB are involved in the down-regulation of NPR-A activity in H9c2 (2-1) cardiac myoblast cells. The H9c2 (2-1) cardiac myoblast cells were exposed to ANG II (10(-7) M for 20 h) with/or without blocker treatment (losartan-10 µM, N-acetyl cysteine (NAC)-10 mM and pyrrolidine dithiocarbamate (PDTC)-100 µM). On exposure, ANG II induced a significant decrease (P < 0.001) in the expression of Npr1 (coding for NPR-A) gene and NPR-A receptor-dependent guanylyl cyclase (GC) activity. The level of expression of proto-oncogenes (c-fos, c-myc, and c-jun) and natriuretic peptides (ANP and BNP) was increased in ANG II-treated cells when compared with control cells. Interestingly, ANG II-dependent repression of Npr1 gene expression and guanylyl cyclase (GC) activity was completely restored on treatment with losartan, while only a partial reversal was observed in NAC- and PDTC-co-treated cells. In conclusion, the results of this study suggest that ROS-mediated NF-κB activation mechanism is critically involved in the ANG II-mediated down-regulation of NPR-A expression and its GC activity.

Triiodo-L-thyronine (T3) downregulates Npr1 gene (coding for natriuretic peptide receptor-A) transcription in H9c2 cells: involvement of ß-AR-ROS signaling.

INTRODUCTION: Natriuretic peptide receptor-A (NPR-A) signaling system is considered as an intrinsic productive mechanism of the heart that opposes abnormal cardiac remodeling and hypertrophic growth. NPR-A is coded by Npr1 gene, and its expression is downregulated in the hypertrophied heart. AIM: We sought to examine the levels of Npr1 gene transcription in triiodo-L-thyronine (T3) treated hypertrophied cardiomyocyte (H9c2) cells, in vitro, and also the involvement of ß-adrenergic receptor (ß-AR) - Reactive oxygen species (ROS) signaling system in the down-regulation of Npr1 transcription also studied. MAIN METHODS: Anti-hypertrophic Npr1 gene transcription was monitored in control and T3-treated (dose and time dependent) H9c2 cells, using a real time PCR method. Further, cell size, intracellular cGMP, ROS, hypertrophy markers (ANP, BNP, α-sk, α-MHC and ß-MHC), ß-AR, and protein kinase cGMP-dependent 1 (PKG-I) genes expression were also determined. The intracellular cGMP and ROS levels were determined by ELISA and DCF dye method, respectively. In addition, to neutralize T3 mediated ROS generation, H9c2 cells were treated with T3 in the presence and absence of antioxidants [curcumin (CU) or N-acetyl-L-cysteine (NAC)]. RESULTS: A dose dependent (10 pM, 100 pM, 1 nM and 10 nM) and time dependent (12 h, 24 h and 48 h) down-regulation of Npr1 gene transcription (20, 39, 60, and 74% respectively; 18, 55, and 85%, respectively) were observed in T3-treated H9c2 cells as compared with control cells. Immunofluorescence analysis also revealed that a marked down regulation of NPR- A protein in T3-treated cells as compared with control cells. Further, a parallel downregulation of cGMP and PKG-I (2.4 fold) were noticed in the T3-treated cells. In contrast, a time dependent increased expression of ß-AR (60, 72, and 80% respectively) and ROS (26, 48, and 74%, respectively) levels were noticed in T3-treated H9c2 cells as compared with control cells. Interestingly, antioxidants, CU or NAC co-treated T3 cells displayed a significant reduction in ROS (69 and 81%, respectively) generation and to increased Npr1 gene transcription (81 and 88%, respectively) as compared with T3 alone treated cells. CONCLUSION: Our result suggest that down regulation of Npr1 gene transcription is critically involved in T3- induced hypertrophic growth in H9c2 cells, and identifies the cross-talk between T3-ß-AR-ROS and NPR-A signaling.

Suppression of atrial natriuretic peptide/natriuretic peptide receptor-A-mediated signaling upregulates angiotensin-II-induced collagen synthesis in adult cardiac fibroblasts.

Cardiac hormone atrial natriuretic peptide (ANP) and its receptor natriuretic peptide receptor-A (NPR-A) system acts as an intrinsic negative regulator of abnormal extracellular matrix (ECM) remodeling in the heart. However, the underlying mechanism by which ANP/NPR-A system opposes the ECM remodeling in the diseased heart is not well understood. Here, we investigated the anti-fibrotic mechanism of ANP/NPR-A in fibrotic agonist Angiotensin- II (ANG II)-treated adult cardiac fibroblast (CF) cells. Normal and NPR-A-suppressed adult CF cells were treated with ANG II (10(-7) M) in the presence and absence of ANP (10(-8) M) for 24 h. Total collagen concentration, activity and expression of MMP-2 and MMP-9, and nuclear translocation of Nuclear factor-kappaB (NF-κB-p50) were studied. NPR-A-suppressed adult CF cells exhibited a more pronounced increase in collagen production, ROS generation, and NF-κB-p50 nuclear translocation as compared to adult CF cells treated with agonist alone. ANP co-treatment significantly reverses the agonist-induced above changes in normal adult CF cells, while it failed to reverse the agonist-induced collagen synthesis in the NPR-A-suppressed adult CF cells. The cGMP analog (8-bromo-cGMP) treatment significantly attenuated the agonist-induced collagen synthesis both in normal and NPR-A-suppressed adult cells. The results of this study suggest that ANP/NPR-A signaling system antagonizes the agonist-induced collagen synthesis via suppressing the activities of MMP-2, MMP-9, ROS generation, and NF-κB nuclear translocation mechanism.

Angiotensin-II down-regulates cardiac natriuretic peptide receptor-A mediated anti-hypertrophic signaling in experimental rat hearts.

Atrial natriuretic peptide (ANP) exerts anti-hypertrophic effects in the heart via natriuretic peptide receptor-A (NPR-A). However, ANP mediated anti-hypertrophic activity is decreased in the cardiomyopathic conditions. In the present investigation the in vivo effects of angiotensin II (Ang II), a hypertrophic agonist have been studied on the ventricular expression level of NPR-A in Wistar rat hearts. NPR-A expression at the protein and mRNA levels were found to be markedly reduced by 5-fold respectively in Ang II infused rats heart as compared with sham rat hearts. Moreover, cGMP production in response to ANP was reduced by 77% in the isolated cardiac membrane preparation from the Ang II infused rat hearts. Losartan treatment reversed NPR-A expression and responsiveness to ANP. This study suggests that Ang II down regulates cardiac NPR-A activity by suppressing Npr1 gene transcription.

Diet-Derived Advanced Glycation End Products (dAGEs) Induce Proinflammatory Cytokine Expression in Cardiac and Renal Tissues of Experimental Mice: Protective Effect of Curcumin.

The beneficial effect of curcumin (CU) on dietary AGEs (dAGEs) involves blocking the overexpression of proinflammatory cytokine genes in the heart and kidney tissues of experimental mice. The animals were divided into six groups (n = 6/group) and were fed a heat-exposed diet (dAGEs) with or without CU for 6 months. Their blood pressure (BP) was monitored by a computerized tail-cuff BP-monitoring system. The mRNA and protein expression levels of proinflammatory genes were analyzed by RT-PCR and western blot, respectively. A marked increase in BP (108 ± 12 mmHg vs 149 ± 15 mmHg) accompanied by a marked increase in the heart and kidney weight ratio was noted in the dAGE-fed mice. Furthermore, the plasma levels of proinflammatory molecules (C5a, ICAM-1, IL-6, MCP-1, IL-1ß and TNF-α) were found to be elevated (3-fold) in dAGE-fed mice. mRNA expression analysis revealed a significant increase in the expression levels of inflammatory markers (Cox-2, iNOS, and NF-κB) (3-fold) in cardiac and renal tissues of dAGE-fed mice. Moreover, increased expression of RAGE and downregulation of AGER-1 (p < 0.001) were noticed in the heart and kidney tissues of dAGE-fed mice. Interestingly, the dAGE-induced proinflammatory genes and inflammatory responses were neutralized upon cotreatment with CU. The present study demonstrates that dietary supplementation with CU has the ability to neutralize dAGE-induced adverse effects and alleviate proinflammatory gene expression in the heart and kidney tissues of experimental mice.

Regulation of cardiac angiotensin-converting enzyme and angiotensin AT1 receptor gene expression in Npr1 gene-disrupted mice.

1. Understanding of the regulatory mechanisms of gene expression in the control of blood pressure and fluid volume is a key issue in cardiovascular medicine. Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signalling antagonizes the physiological and pathophysiological effects mediated by the renin-angiotensin-aldosterone system (RAAS) in the regulation of cardiovascular homeostasis. 2. The targeted-disruption of the Npr1 gene (coding for GC-A/PRA) leads to activation of the cardiac RAAS involved in the hypertrophic remodelling process, which influences cardiac size, expression of pro-inflammatory cytokine genes and the behaviour of various hypertrophy marker genes. The Npr1 gene-knockout (Npr1(-/-)) mice exhibit 35-40 mmHg higher systolic blood pressure and a significantly greater heart weight to bodyweight ratio than wild-type (Npr1(+/+)) mice. 3. The expression of both angiotensin-converting enzyme (ACE) and angiotensin II AT(1a) receptors are significantly increased in hearts from Npr1(-/-) mice compared with hearts from Npr1(+/+) mice. In parallel, the expression of interleukin-6 and tumour necrosis factor-alpha is also markedly increased in hearts from Npr1(-/-) mice. 4. These findings indicate that disruption of NPRA/cGMP signalling leads to augmented expression of the cardiac RAAS in conjunction with pro-inflammatory cytokines in Npr1-null mutant mice, which promotes the development of cardiac hypertrophy and remodelling.

Divergent Synthesis and Evaluation of the in†vitro Cytotoxicity Profiles of 3,4-Ethylenedioxythiophenyl-2-propen-1-one Analogues.

A new series of 3,4-ethylenedioxythiophene (EDOT)-appended propenones were prepared by condensation reaction and their in vitro cytotoxicity effects were evaluated against five human cancer cell lines. Preliminary structure-activity relationships of EDOT-incorporated 2-propenone derivatives were also established. The EDOT-appended enones demonstrated significant cytotoxicity against human cancer cell lines. The most active analogue, (E)-3-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3 p, GI50 =110 nm), severely inhibited the clonogenic potential of cancer cells, and induced cell-cycle arrest in the G2/M phase and caused an accumulation of HCT116 colon cancer cells with >4 N DNA content. Also, 3 p exhibited weak inhibition of the enzymatic activity of human topoisomerase I. Molecular docking studies indicated preferential binding of the compounds to the ATP-binding pocket of the human checkpoint 2 kinase (Chk2) catalytic domain, thus, identifying a novel diaryl 2-propenone chemotype for the development of potent inhibitors of Chk2.

Diet with high content of advanced glycation end products induces systemic inflammation and weight gain in experimental mice: Protective role of curcumin and gallic acid.

The present study was aimed to investigate the effect of diet derived AGEs (dAGEs) on the circulatory levels of pro-inflammatory cytokines, chemokines and to evaluate the protective efficacy of natural anti-oxidants curcumin (CU) and gallic acid (GA) respectively against the dAGEs-induced systemic inflammation in experimental Swiss albino mice. The experimental mice were fed with dAGEs in the presence and absence of CU and GA for 6 months. The levels of 40 circulatory pro-inflammatory cytokines and chemokines were evaluated using Proteome-Cytokine Array kit. In addition, serum levels of N-ɛCML, CRP and HbA1c were estimated by ELISA method. Among the sixteen pro- and anti-inflammatory cytokines analysed, five (IL-16, IL-1α, ICAM, TIMP-1 and C5a) were found to be highly expressed (3.5-fold) and eleven cytokines were moderately expressed (2-fold) in dAGEs fed mice. In case of chemokines, three (BLC, SDF-1 and MCP-1) were found to be highly expressed (4-fold) and ten showed moderate expression (2-fold) as compared with basal diet fed mice. Interestingly, CU or GA co-treatment normalized the levels of circulatory pro- and anti-inflammatory cytokines, chemokines, N-ɛCML, CRP and HbA1c levels. Together, the present study suggests that dAGEs are positively associated with the development of systemic inflammation in experimental mice.

Novel isothiacalothrixin B analogues exhibit cytotoxic activity on human colon cancer cells in vitro by inducing irreversible DNA damage.

Preliminary cytotoxic analysis of sulphur containing isosteric analogues of calothrixin B identified the useful anti-tumour activity of thia/isothiacalothrixin B which necessitated it's biological evaluation in colon and lung cancer cell lines. The isothia analogues induced cytotoxicity of HCT116 in a time-dependent manner and inhibited the clonogenic survival of HCT116 and NCI-H460 cells in a dose-dependent manner comparable to the standard anti-cancer drug camptothecin. Herein employing flow cytometry, we demonstrate that isothiacalothrixin B analogues inhibited proliferation of colon cancer cells by the arrest of cells in S and G2/M phases over a period of 48 hours at a concentration of 5 µM. Our results also suggest that the cytotoxicity of thia analogues of calothrixin B is partially mediated by induction of cellular DNA strand breaks. The UV-Vis spectroscopic studies with CT-DNA revealed groove binding for calothrixin B and its thia analogues wherein subsequent in silico molecular modelling studies indicated preferential binding to the AT-rich regions of minor groove of DNA. Furthermore, thiacalothrixin B caused transcriptional activation of p21waf1/cip1 promoter and upregulation of its protein levels independent of p53. The induction of DNA damage response pathway leads to apoptosis in isothiacalothrixin B but not in thiacalothrixin B treated cells. The isothia analogues SCAB 4 induced DNA strand breaks and cell cycle arrest even after treatment for a short period (i.e., 4 hours) and the cell cycle effects were irreversible. For the first time, this study provides detailed cellular effects on the potential use of isothiacalothrixin B analogues as cytotoxic agents.

Synthesis and Biological Evaluation of Calothrixins B and their Deoxygenated Analogues.

A series of calothrixin B (2) analogues bearing substituents at the 'E' ring and their corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The cytotoxicities of calothrixins 1, 2, and 15b-p and quinocarbazole analogues were investigated against nine cancer cell lines. The quinocarbazoles 21a and 25a inhibited the catalytic activity of human topoisomerase II. The plasmid DNA cleavage abilities of calothrixins 1, 2, and 15b-p identified compound 15h causing DNA cleavage comparable to that of calothrixin A (1). Calothrixin A (1), 3-fluorocalothrixin 15h and 4-fluoroquinocarbazole 21b induced extensive DNA damage followed by apoptotic cell death. Spectral and plasmid unwinding studies demonstrated an intercalative mode of binding for quinocarbazoles. We identified two promising drug candidates, the 3-fluorocalothrixin B 15h with low toxicity in animal model and its deoxygenated derivative 4-fluoroquinocarbazole 21b as having potent cytotoxicity against NCI-H460 cell line with a GI50 of 1 nM.

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A upregulates ACE and AT1 receptor gene expression and signaling: role in cardiac hypertrophy.

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) signaling antagonizes the physiological effects mediated by the renin-angiotensin system (RAS). The objective of this study was to determine whether the targeted-disruption of Npr1 gene (coding for GC-A/NPRA) leads to the activation of cardiac RAS genes involved on the hypertrophic remodeling process. The Npr1 gene-knockout (Npr1(-/-)) mice showed 30-35 mmHg higher systolic blood pressure (SBP) and a 63% greater heart weight-to-body weight (HW/BW) ratio compared with wild-type (Npr1(+/+)) mice. The mRNA levels of both angiotensin-converting enzyme and angiotensin II type 1a receptor were increased by three- and fourfold, respectively, in Npr1(-/-) null mutant mice hearts compared with the wild-type Npr1(+/+) mice hearts. In parallel, the expression levels of interleukin-6 and tumor necrosis factor-alpha were increased by four- to fivefold, in Npr1(-/-) mice hearts compared with control animals. The NF-kappaB binding activity in nuclear extracts of Npr1(-/-) mice hearts was increased by fourfold compared with wild-type Npr1(+/+) mice hearts. Treatments with captopril or hydralazine equally attenuated SBP; however, only captopril significantly decreased the HW/BW ratio and suppressed cytokine gene expression in Npr1(-/-) mice hearts. The ventricular cGMP level was reduced by almost sixfold in Npr1(-/-) mice compared with wild-type control mice. The results of the present study indicate that disruption of NPRA/cGMP signaling leads to the augmented expression of cardiac RAS pathways that promote the development of cardiac hypertrophy and remodeling.

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A.

Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1(-/-)) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1(+/+)) mice. A significant up-regulation of cytokine genes such as TNF-alpha (five-fold), IL-6 (three-fold) and TGF-beta1 (four-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-kappaB binding activity was almost five-fold greater in the nuclear extract of Npr1(-/-) mutant mice hearts as compared with wild-type Npr1(+/+) mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10- and 5-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-kappaB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.

A novel nano-hydroxyapatite - PMMA hybrid scaffolds adopted by conjugated thermal induced phase separation (TIPS) and wet-chemical approach: Analysis of its mechanical and biological properties.

In this study, we report the preparation of nano-hydroxyapatite (nHAp) incorporated poly(methyl methacrylate) (PMMA) scaffolds by conjugated thermal induced phase separation (TIPS) and wet-chemical approach, which essentially facilitates the enhancement of both mechanical as well as biological properties of the scaffolds. The dissolution of PMMA was accomplished by acetone (Ace scaffold), ethanol-water (E-W scaffold) and isopropanol-water (I-W scaffold) mixtures as solvents. The existence of nHAp in PMMA matrix was investigated systematically. The higher degree of porous architecture was achieved from Ace scaffolds compared to both I-W and E-W scaffolds. On the other hand, the dense porous architecture of the I-W scaffold exhibited superior hardness and compressive strength than that of the Ace and E-W scaffolds. All the fabricated samples demonstrated enhanced in vitro bioactivity with respect to increasing immersion period as a result of flower-like in vitro apatite layer formation. The MTT assay was carried out for 1day and 3day culture using Saos-2 osteoblast-like cells, which showed better cell proliferation with increasing culture period owing to the interconnected pore architecture of scaffolds and the rational hemocompatibility as per the ASTM standard F756-00.

WITHDRAWN: A novel nano-hydroxyapatite - PMMA hybrid scaffolds adopted by conjugated thermal induced phase separation (TIPS) and wet-chemical approach: Analysis of its mechanical and biological properties.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Mater. Sci. Eng.: C, 73 (2017) 164­172, 10.1016/http://dx.doi.org/j.msec.2016.12.133. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Valporic acid enhances the Atrial Natriuretic Peptide (ANP) mediated anti-hypertrophic activity by modulating the Npr1 gene transcription in H9c2 cells in vitro.

The present study was aimed to determine whether stimulating Npr1 gene activity using Valporic acid (VA), a small short chain fatty acid molecule can enhance ANP mediated anti-hypertrophic activity in isoproterenol (ISO) - treated H9c2 cells in vitro. H9c2 cells were treated with ISO (10-5 M) and co-treated with VA (10-5 M) in the presence and absence of ANP (10-8M), for 48h. ATRA (10-5 M) was used as a positive inducer of Npr1 gene transcription. The mRNA expression of Npr1 and PKG-I genes, proto-oncogenes (c-fos, c-jun and c-myc) and hypertrophic markers (ANP, BNP, α-sk and ß-MyHC), genes were determined by quantitative PCR (qPCR). The protein profiling of NPR-A, PKG-I and cGMP were evaluated by Western blot, immunofluorescence and ELISA respectively. A marked reduction in the level of expression of Npr1 (3- fold) and PKG-I (2.5-fold) genes and increased expression of proto-oncogenes (p< 0.001, respectively) and hypertrophic marker genes (p<0.001, respectively) were noticed in the ISO-treated H9c2 cells as compared with control cells. In contrast, the VA treated cells showed maximal Npr1 gene expression (3.5-fold) as compared with ATRA treated cells (2 fold), which is well correlated with the intracellular cGMP levels (80% vs 60%) and reduced (2.5-fold) HDAC -1&-2 mRNA expression. Furthermore, VA or ATRA treatment effectively reversed the ISO-induced altered expression of Npr1 and PKG-I genes, proto-oncogenes, and hypertrophic markers genes. Interestingly, the results of the present study suggest that ANP mediated anti-hypertrophic activity was enhanced with either VA (p<0.001) or ATRA (p<0.01) co-treatment. Together, we conclude that VA in combination with ANP can be a novel therapeutical approach for the treatment and management of left ventricular cardiac hypertrophy.

Data on synthesis and characterization of chitosan nanoparticles for in vivo delivery of siRNA-Npr3: Targeting NPR-C expression in the heart.

This data article contains the data related to the research article 'Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated ß-adrenoceptor activation-induced cardiac hypertrophic growth in experimental rats' (Venkatesan et al., 2016 [1]). The siRNA-Npr3 loaded chitosan nanoparticles were synthesized using ionotropic gelation method, where the positive charge of the chitosan interacts with the negative charge of STPP and siRNA-Npr3. The physicochemical properties of the synthesized siRNA-Npr3 loaded chitosan nanoparticles were studied by dynamic light scattering, FE-SEM and HR-TEM analysis. In addition, the loading efficiency and stability of the nanoparticles were also studied. Further, the gene silencing efficacy, hemocompatibility and biocompatibility were studied using Wistar rats (in vivo), isolated red blood cells and H9c2 cardiomyoblast cells, respectively.

Differential expression and regulation of anti-hypertrophic genes Npr1 and Npr2 during ß-adrenergic receptor activation-induced hypertrophic growth in rats.

We sought to determine the effect of chronic activation of ß-adrenergic receptor (ß-AR) on the left ventricular (LV) expression profile of Npr1 and Npr2 (coding for NPR-A and NPR-B, respectively) genes, and the functional activity of these receptors in adult Wistar rat hearts. The Npr1 gene expression was markedly reduced (3.5-fold), while the Npr2 gene expression was up regulated (4-fold) in Isoproterenol (ISO)-treated heart as compared with controls. A gradual reduction in NPR-A protein (3-fold), cGMP levels (75%) and a steady increased expression of NPR-B protein (4-fold), were noticed in ISO hearts. Further, in-vitro membranes assay shows that NPR-A dependent guanylyl cyclase (GC) activity was down-regulated (2-fold), whereas NPR-B dependent GC activity was increased (5-fold) in ISO treated hearts. Atenolol treatment normalized the altered expression of Npr1 and Npr2 genes. In conclusion, the chronic ß-AR activation differentially regulates Npr1 and Npr2 genes in the heart. Npr1 down regulation is positively associated with the development of left ventricular hypertrophy (LVH) in ISO rats.

Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated ß-adrenoceptor activation- induced cardiac hypertrophic growth in experimental rats.

Natriuretic peptide receptor-C (NPR-C) is considered as a clearance receptor that maintains the circulatory levels of natriuretic peptides. It has been suggested that augmented expression of NPR-C as a cause for the diminished anti-hypertrophic action of natriuretic peptides in the failing heart. Hence, we sought to determine the level of Npr3 gene (coding for NPR-C) expression in the Isoproterenol (ISO) treated Wistar rats. In addition, we studied the effect of Npr3 gene silencing on the hypertrophic growth. A significant increase in heart weight-to-body weight ratio (HW/BW-24%,P<0.01), an indicator of cardiac hypertrophic growth was observed in the ISO (10mg/kg BW/day,i.p for 7 days) treated rats. As expected, the cardiac NPR-C protein expression was significantly increased by 4 fold as compared to control rats. In parallel, the circulatory atrial natriuretic peptide (ANP) level was significantly decreased (2 fold) in ISO treated rats. Upon treatment with siRNA-Npr3, a significant decrease in the cardiac NPR-C protein expression (70%,P<0.01), HW/BW ratio (70%,P<0.01) and hypertrophic marker genes (α-Sk, ß-MHC, c-fos, P<0.01, respectively) mRNA expression were observed. Interestingly, the circulatory ANP level was increased by 1.5 fold in the siRNA-Npr3 treated rats as compared to ISO treated rats. Moreover, the cardiac collagen content, matrixmetalloprotinases-2 (MMP-2) and enzymatic antioxidant status (P<0.01, respectively) were found to be restored back to near normal upon siRNA-Npr3 treatment. Taken together, the results of this study indicates that specific down-regulation of Npr3 gene improves the circulatory levels of ANP and antioxidant system and there by attenuates the ß-adrenoceptor over-activation mediated cardiac hypertrophic growth in experimental rats.