Deep Transcriptomic Profiling of M1 Macrophages Lacking Trpc3

In previous studies using mice with macrophage-specific loss of TRPC3 we found a significant, selective effect of TRPC3 on the biology of M1, or inflammatory macrophages. Whereas activation of some components of the unfolded protein response and the pro-apoptotic mediators CamkII and Stat1 was impaired in Trpc3-deficient M1 cells, gathering insight about other molecular signatures within macrophages that might be affected by Trpc3 expression requires an alternative approach. In the present study we conducted RNA-seq analysis to interrogate the transcriptome of M1 macrophages derived from mice with macrophage-specific loss of TRPC3 and their littermate controls. We identified 160 significantly differentially expressed genes between the two groups, of which 62 were upregulated and 98 downregulated in control vs. Trpc3-deficient M1 macrophages. Gene ontology analysis revealed enrichment in processes associated to cellular movement and lipid signaling, whereas the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included networks for calcium signaling and cell adhesion molecules, among others. This is the first deep transcriptomic analysis of macrophages in the context of Trpc3 deficiency and the data presented constitutes a unique resource to further explore functions of TRPC3 in macrophage biology.Fil: Kumarasamy, Sivarajan. University of Toledo; Estados UnidosFil: Solanki, Sumeet. University of Toledo; Estados UnidosFil: Atolagbe, Oluwatomisin T.. University of Toledo; Estados UnidosFil: Joe, Bina. University of Toledo; Estados UnidosFil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Vazquez, Guillermo. University of Toledo; Estados Unido

Dysglycemia induces abnormal circadian blood pressure variability

<p>Abstract</p> <p>Background</p> <p>Prediabetes (PreDM) in asymptomatic adults is associated with abnormal circadian blood pressure variability (abnormal CBPV).</p> <p>Hypothesis</p> <p>Systemic inflammation and glycemia influence circadian blood pressure variability.</p> <p>Methods</p> <p>Dahl salt-sensitive (S) rats (n = 19) after weaning were fed either an American (AD) or a standard (SD) diet. The AD (high-glycemic-index, high-fat) simulated customary human diet, provided daily overabundant calories which over time lead to body weight gain. The SD (low-glycemic-index, low-fat) mirrored desirable balanced human diet for maintaining body weight. Body weight and serum concentrations for fasting glucose (FG), adipokines (leptin and adiponectin), and proinflammatory cytokines [monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α)] were measured. Rats were surgically implanted with C40 transmitters and blood pressure (BP-both systolic; SBP and diastolic; DBP) and heart rate (HR) were recorded by telemetry every 5 minutes during both sleep (day) and active (night) periods. Pulse pressure (PP) was calculated (PP = SBP-DBP).</p> <p>Results</p> <p>[mean(SEM)]: The AD fed group displayed significant increase in body weight (after 90 days; p < 0.01). Fasting glucose, adipokine (leptin and adiponectin) concentrations significantly increased (at 90 and 172 days; all p < 0.05), along with a trend for increased concentrations of systemic pro-inflammatory cytokines (MCP-1 and TNF-α) on day 90. The AD fed group, with significantly higher FG, also exhibited significantly elevated circadian (24-hour) overall mean SBP, DBP, PP and HR (all p < 0.05).</p> <p>Conclusion</p> <p>These data validate our stated hypothesis that systemic inflammation and glycemia influence circadian blood pressure variability. This study, for the first time, demonstrates a cause and effect relationship between caloric excess, enhanced systemic inflammation, dysglycemia, loss of blood pressure control and abnormal CBPV. Our results provide the fundamental basis for examining the relationship between dysglycemia and perturbation of the underlying mechanisms (adipose tissue dysfunction induced local and systemic inflammation, insulin resistance and alteration of adipose tissue precursors for the renin-aldosterone-angiotensin system) which generate abnormal CBPV.</p

SAR and molecular mechanism studies of monoamine oxidase inhibition by selected chalcone analogs

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC50 values were found to be in the micromolar to submicromolar range. The Ki values of compound 16 were determined to be 0.047 and 0.020 μM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme.This work was supported by internal grants from the Office of Academic Research, Qatar University, Doha, Qatar under Grants [# QUUG-CPH-15/16–7 and QUUG-CPH-2017–5]. The publication of this article was funded by the Qatar National Library, Doha, Qatar. The experiments dealing with the testing of the compounds in vitro were supported by the National Institutes on Alcohol Abuse and Alcoholism under Grant Number R01AA019458 (Y.S.). We are grateful to Professor Neal Castagnoli Jr. Professor Emeritus, Virginia Tech for providing the MMTP substrate. The biological section of this study was funded in part by the National Research Foundation of South Africa under Grant [number (UID) 85642, 96180]. The Grantholders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by the NRF supported research are that of the authors and that the NRF accepts no liability whatsoever in this regard.Scopu

Mitochondrial polymorphisms in rat genetic models of hypertension

Hypertension is a complex trait that has been studied extensively for genetic contributions of the nuclear genome. We examined mitochondrial genomes of the hypertensive strains: the Dahl Salt-Sensitive (S) rat, the Spontaneously Hypertensive Rat (SHR), and the Albino Surgery (AS) rat, and the relatively normotensive strains: the Dahl Salt-Resistant (R) rat, the Milan Normotensive Strain (MNS), and the Lewis rat (LEW). These strains were used previously for linkage analysis for blood pressure (BP) in our laboratory. The results provide evidence to suggest that variations in the mitochondrial genome do not account for observed differences in blood pressure between the S and R rats. However, variants were detected among the mitochondrial genomes of the various hypertensive strains, S, SHR, and AS, and also among the normotensive strains R, MNS, and LEW. A total of 115, 114, 106, 106, and 16 variations in mtDNA were observed between the comparisons S versus LEW, S versus MNS, S versus SHR, S versus AS, and SHR versus AS, respectively. Among the 13 genes coding for proteins of the electron transport chain, 8 genes had nonsynonymous variations between S, LEW, MNS, SHR, and AS. The lack of any sequence variants between the mitochondrial genomes of S and R rats provides conclusive evidence that divergence in blood pressure between these two inbred strains is exclusively programmed through their nuclear genomes. The variations detected among the various hypertensive strains provides the basis to construct conplastic strains and further evaluate the effects of these variants on hypertension and associated phenotypes

Deep transcriptomic profiling of Dahl salt-sensitive rat kidneys with mutant form of Resp18.

Expression of Regulated endocrine specific protein 18 (Resp18) is localized in numerous tissues and cell types; however, its exact cellular function is unknown. We previously showed that targeted disruption of the Resp18 locus in the Dahl SS (SS) rat (Resp18(mutant)) results in higher blood pressure (BP), increased renal fibrosis, increased urinary protein excretion, and decreased mean survival time following a chronic (6 weeks) 2% high salt (HS) diet compared with the SS rat. Based on this prominent renal injury phenotype, we hypothesized that targeted disruption of Resp18 in the SS rat promotes an early onset hypertensive-signaling event through altered signatures of the renal transcriptome in response to HS. To test this hypothesis, both SS and Resp18(mutant) rats were exposed to a 7-day 2% HS diet and BP was recorded by radiotelemetry. After a 7-day exposure to the HS diet, systolic BP was significantly increased in the Resp18(mutant) rat compared with the SS rat throughout the circadian cycle. Therefore, we sought to investigate the renal transcriptomic response to HS in the Resp18(mutant) rat. Using RNA sequencing, Resp18(mutant) rats showed a differential expression of 25 renal genes, including upregulation of Ren. Upregulation of renal Ren and other differentially expressed genes were confirmed via qRT-PCR. Moreover, circulating renin activity was significantly higher in the Resp18(mutant) rat compared with the WT SS rat after 7 days on HS. Collectively, these observations demonstrate that disruption of the Resp18 gene in the SS rat is associated with an altered renal transcriptomics signature as an early response to salt load

Characterization of a Long Non-Coding RNA, the Antisense RNA of Na/K-ATPase α1 in Human Kidney Cells

Non-coding RNAs are important regulators of protein-coding genes. The current study characterized an antisense long non-coding RNA, ATP1A1-AS1, which is located on the opposite strand of the Na/K-ATPase &alpha;1 gene. Our results show that four splice variants are expressed in human adult kidney cells (HK2 cells) and embryonic kidney cells (HEK293 cells). These variants can be detected in both cytosol and nuclear fractions. We also found that the inhibition of DNA methylation has a differential effect on the expression of ATP1A1-AS1 and its sense gene. To investigate the physiological role of this antisense gene, we overexpressed the ATP1A1-AS1 transcripts, and examined their effect on Na/K-ATPase expression and related signaling function in human kidney cells. The results showed that overexpression of the ATP1A1-AS1-203 transcript in HK2 cells reduced the Na/K-ATPase &alpha;1 (ATP1A1) gene expression by approximately 20% (p &lt; 0.05), while reducing the Na/K-ATPase &alpha;1 protein synthesis by approximately 22% (p &lt; 0.05). Importantly, overexpression of the antisense RNA transcript attenuated ouabain-induced Src activation in HK2 cells. It also inhibited the cell proliferation and potentiated ouabain-induced cell death. These results demonstrate that the ATP1A1-AS1 gene is a moderate negative regulator of Na/K-ATPase &alpha;1, and can modulate Na/K-ATPase-related signaling pathways in human kidney cells

SAR and molecular mechanism studies of monoamine oxidase inhibition by selected chalcone analogs

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC50 values were found to be in the micromolar to submicromolar range. The Ki values of compound 16 were determined to be 0.047 and 0.020 μM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme