Caloric restriction improves inflammation in different tissues of the Wistar rats with obesity and 2K1C renovascular hypertension.

Renovascular hypertension (RHV) is the cause of high blood pressure due to left renal ischemia, and obesity and hypertension cause an inflammatory response. This work analyzed the inflammatory and tissue repair profile in renal, hepatic, and cardiac tissues in an animal model of RVH associated with a high-fat diet and caloric restriction. The expressions of RORγ-t, IL-17, T-bet, and TNF-α decreased and IFN-γ increased in the right kidney. In relation to the left kidney, caloric restriction decreased the expression of IFN-γ. In the liver, caloric restriction decreased RORγ-t, IL-17, and T-bet. Hypertension associated with obesity decreased the expression of IFN-γ, while caloric restriction increased. In the right kidney, hypertension and obesity, associated or not with caloric restriction, increased the area of collagen fibers. In the heart and liver, caloric restriction reduced the area of collagen fibers. Caloric restriction increased vascular endothelial growth factor, reduced levels of growth transformation factor-ß1 (TGF-ß), and increased collagen I in the left kidney. Hypertension/obesity, submitted or not having caloric restriction, increased TGF-ß in liver. The results suggest that caloric restriction has beneficial effects in lowering blood pressure and regulating tissue proinflammatory cytokines. However, there was no change in the structure and composition of tissue repair markers.

Absence of melatonin during development impairs craniofacial and dental onset in rats.

OBJECTIVE: Herein, we evaluated pinealectomy-induced melatonin absence to determine its effects on craniofacial and dental development in the offspring. DESIGN: Female Wistar rats in three groups, i.e., intact pregnant rats, pinealectomized pregnant rats (PINX), and pinealectomized pregnant rats subjected to oral melatonin replacement therapy, were crossed 30 days after surgery. The heads of 7-day-old pups were harvested for cephalometric and histological analyses, and maxillae and incisors were collected for mRNA expression analysis. RESULTS: The PINX pups exhibited a reduction in neurocranial and facial parameters such as a decrease in alveolar bone area, incisor size and proliferation, and an increase in odontoblasts and the dentin layer. Based on incisor mRNA expression analysis, we found that Dmp1 expression was upregulated, whereas Col1a1 expression was downregulated. Maxillary mRNA expression revealed that Rankl expression was upregulated, whereas that of Opn and Osx was downregulated. CONCLUSION: Our results demonstrated that the absence of maternal melatonin during early life could affect dental and maxillary development in offspring, as well as delay odontogenesis and osteogenesis in maxillary tissues. CLINICAL RELEVANCE: Our findings suggest that disruptions or a lack of melatonin during pregnancy may cause changes in craniofacial and dental development, at least in animal experiments; however, in humans, these feedings are still poorly understood, and thus careful evaluations of melatonin levels in humans need to be investigated in craniofacial alterations.

Maxillary and dental development in the offspring of protein-restricted female rats.

Nutritional restriction during developmental periods impairs organ physiology. Female rats were subjected to protein restriction during pregnancy and lactation to analyze dental and maxillary development. Four exposure groups were considered: normal-protein diet during pregnancy and lactation (NP, 17% casein), low-protein diet during lactation (LP-L, 6% casein), low-protein diet during pregnancy and lactation (LP), and low-protein diet during pregnancy (LP-G). Maxillae from 15-day-old male pups were collected. All protein-restricted groups presented increased dentin thickness and reduced alveolar bone area. When protein restriction was applied during both gestation and lactation (LP), harmful effects were observed in the form of loss of protective OPG (osteoprotegerin) in tooth epithelium-mesenchyme, due to higher RANKL expression, delay in odontoblast maturation, less dental pulp vascularity, reduction in amount of alveolar bone, and less matrix mineralization. In the LP-L group, effects of protein restriction seemed less harmful, and despite less alveolar bone, the enhancement in BMP-7, VEGF, and RANKL seems a compensatory signal to maintain maxillary osteogenesis. In LP-G animals, Dspp expression was higher, suggesting a delay in odontoblast maturation or expression recuperation. In conclusion, maternal protein restriction affects dental and maxillary development. A low-protein diet only in gestation allows for normal development. A low-protein diet during gestation-lactation results in impaired odontogenesis that may increase susceptibility of dental anomalies.

Quercetin prevents insulin dysfunction in hypertensive animals.

Angiotensin II induced increase in hypertension enhances oxidative stress and compromises insulin action and pancreatic function. Quercetin-rich foods are beneficial for hypertensive and diabetic animals owing to their antioxidant function. The aim of this study was to evaluate the antioxidant effects of quercetin in hypertensive rats on insulin action, signaling, and secretion. Wistar rats were randomly divided into three groups: sham, hypertensive rats (H), and hypertensive rats supplemented with quercetin (HQ). After three months of initial hypertension, quercetin was administered at 50 mg/kg/day for 30 days. Our results indicate that hypertension and serum lipid peroxidation levels were reduced by quercetin supplementation. We observed increased insulin sensitivity in adipose tissue, corroborating the insulin tolerance test, HOMA index, and improvements in lipid profile. Despite normal insulin secretion at 2.8 and 20 mM of glucose, animals treated with quercetin exhibited increased number of islets per section; increased protein expression of muscarinic receptor type 3, VEGF, and catalase in islets; and hepatic mRNA levels of Ide were normalized. In conclusion, supplementation with quercetin improved insulin action and prevented pancreatic and metabolic dysfunction.

Caloric restriction overcomes pre-diabetes and hypertension induced by a high fat diet and renal artery stenosis.

BACKGROUND: Calorie restriction (CR) is a type of dietary intervention that is essential in weight loss through modulation of critical metabolic control pathways, is well established and understood in cases of systemic arterial hypertension, however, its role in renovascular hypertension is still unclear. METHODS: Rats were divided into three groups: SHAM, and two groups that underwent surgery to clip the left renal artery and induce renovascular hypertension (OH and OHR). The SHAM diet was as follows: 14 weeks normolipidic diet; OH: 2 weeks normolipidic diet + 12 weeks hyperlipidic diet, both ad libitum; OHR, 2 weeks normolipidic diet + 8 weeks ad libitum high-fat diet + 4 weeks 40% calorie-restricted high-fat diet. RESULTS: Rats in the OHR group had decreased blood pressure, body weight, and glucose levels. Reductions in insulinemia and in lipid and islet fibrotic areas in the OHR group were observed, along with increased insulin sensitivity and normalization of insulin-degrading enzyme levels. The expression of nicotinamide phosphoribosyltransferase (NAMPT), insulin receptor (IR), sirtuin 1 (SIRT1), and complex II proteins were increased in the liver tissue of the OHR group. Strong correlations, whether positive or negative, were evaluated via Spearman's model between SIRT1, AMPK, NAMPT, PGC-1α, and NNMT expressions with the restoration of normal blood pressure, weight loss, glycemic and lipid panel, and mitochondrial adaptation. CONCLUSION: CR provided short-term beneficial effects to recover the physiological parameters induced by a high-fat diet and renal artery stenosis in obese and hypertensive animals. These benefits, even in the short term, can provide physiological benefits in the long term.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Regulation of connexins genes expression contributes to reestablishes tissue homeostasis in a renovascular hypertension model.

Connexins (Cx) are essential for cardiovascular regulation and maintenance of cardio-renal response involving the natriuretic peptide family. Changes in the expression of connexins promote intercellular communication dysfunction and may induce hypertension, atherosclerosis, and several other vascular diseases. This study analyzed the expression of the genes involved in the renin-angiotensin system (RAS) and the relation of the connexins gene expression with the renovascular hypertension 2K1C in different tissues. The insertion of a silver clip induced renovascular hypertension 2K1C into the left renal artery. Biochemical measurements were made using commercial kits. Gene expression was evaluated in the liver, heart, and kidneys by RT-PCR. The genes investigated were LDLr, Hmgcr, Agt, Ren, Ace, Agtr1a, Anp, Bnp, Npr1, Cx26, Cx32, Cx37, Cx40 and Cx43. All genes involved in the RAS presented increased transcriptional levels in the 2K1C group, except hepatic Agt. The natriuretic peptides (Anp; Bnp) and the receptor genes (Npr1) appeared to increase in the heart, however, Npr1 decreased in the kidneys. In hepatic tissue, hypertension promoted increased expression of Cx32, Cx37, and Cx40 genes however, Cx26 and Cx43 genes were not influenced. Expression was upregulated for Cx37 and Cx43 in cardiac tissue in the 2K1C group, but Cx40 did not demonstrate any difference between groups. The stenotic kidney showed an upregulated expression for Cx37 vs Sham and contralateral kidney, although Cx40 and Cx43 were downregulated. Hypertension did not modify the transcriptional expression of Cx26 and Cx32. Therefore, this study indicated that RAS and cardiac response were regulated transcriptionally by renovascular hypertension 2K1C. Moreover, the results of connexin gene expression demonstrated differential transcriptional regulation in different tissues studied and suggest a relationship between cardiac and renal physiological changes as an adaptive mechanism to the hypertensive state.

Inhibitory effect of red LED irradiation on fibroblasts and co-culture of adipose-derived mesenchymal stem cells.

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-ß1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-ß1, whereas TGF-ß1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-ß1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.

Tissue-specific transcriptional regulation of epithelial/endothelial and mesenchymal markers during renovascular hypertension.

Epithelial-to-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition are processes that can occur under different biological conditions, including tissue healing due to hypertension and oxidative stress. The purpose of the present study was to evaluate the differences in gene expression of epithelial/endothelial and mesenchymal markers in different tissues. A two-kidney, one-clip (2K1C) renovascular hypertension rat model was used. Hypertension was induced by the clipping of the left renal artery; the rats were randomized into sham and 2K1C groups and monitored for up to 4 weeks. The gene expressions of E-cadherin (E-cad), N-cadherin (N-cad), α-smooth muscle actin (α-SMA), collagen I (COL1A1), collagen III (COL3A1) and hepatocyte growth factor (HGF) were determined by reverse transcription-PCR. The levels of the cytokines transforming growth factor-ß1, tumor necrosis factor-α, interleukin (IL)-4, IL-6 and IL-10 were evaluated using ELISAs. The levels of thiobarbituric acid reactive substances and thiol groups were measured to evaluate oxidative stress. All analyses were performed on the liver, heart and kidneys tissues of sham and model rats. The 2K1C animals exhibited a higher systolic blood pressure, as well as cardiac hypertrophy and atrophy of the left kidney. Fibrotic alterations in the heart and kidneys were observed, as was an increase in the collagen fiber areas, and higher levels of inflammatory cytokines, which are associated with the increased expression of fibroproliferative and anti-fibrotic genes. Renovascular hypertension regulated epithelial/endothelial and mesenchymal markers, including E-cad, N-cad, α-SMA and COL1A1 in the kidneys and heart. EMT in the kidneys was mediated by an increased level of inflammatory and profibrotic cytokines, as well as by oxidative stress. The data in the present study suggested that the expression of epithelial/endothelial and mesenchymal markers are differentially regulated by hypertension in the liver, heart and kidneys.

Single nucleotide polymorphisms of the GJB2 and GJB6 genes are associated with autosomal recessive nonsyndromic hearing loss.

Single nucleotide polymorphisms (SNPs) are important markers in many studies that link DNA sequence variations to phenotypic changes; such studies are expected to advance the understanding of human physiology and elucidate the molecular basis of diseases. The DFNB1 locus, which contains the GJB2 and GJB6 genes, plays a key role in nonsyndromic hearing loss. Previous studies have identified important mutations in this locus, but the contribution of SNPs in the genes has not yet been much investigated. The aim of this study was to investigate the association of nine polymorphisms located within the DFNB1 locus with the occurrence of autosomal recessive nonsyndromic hearing loss (ARNSHL). The SNPs rs3751385 (C/T), rs7994748 (C/T), rs7329857 (C/T), rs7987302 (G/A), rs7322538 (G/A), rs9315400 (C/T), rs877098 (C/T), rs945369 (A/C), and rs7333214 (T/G) were genotyped in 122 deaf patients and 132 healthy controls using allele-specific PCR. There were statistically significant differences between patients and controls, in terms of allelic frequencies in the SNPs rs3751385, rs7994748, rs7329857, rs7987302, rs945369, and rs7333214 (P < 0.05). No significant differences between the two groups were observed for rs7322538, rs9315400, and rs877098. Our results suggest that SNPs present in the GJB2 and GJB6 genes may have an influence on ARNSHL in humans.

Guidelines for the tetra-primer ARMS-PCR technique development.

The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS-PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl2. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS-PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.

Connexin mutations in Brazilian patients with skin disorders with or without hearing loss.

The connexins are a family of proteins whose major function is as part of the gap junctions of cell-to-cell channels. They are expressed in several tissues including brain, skin, and cochlea. Mutations in connexin genes play a major role in non-syndromic sensorineural deafness, but have been also described in individuals with variable dermatological features. In recent years, many genes responsible for hereditary skin diseases have been discovered. These genes may encode different proteins that participate in the terminal differentiation of the epidermis. Therefore alteration or absence of these proteins causes a keratinization disorder. It has been demonstrated that distinct germline mutations within six connexin (Cx) genes GJB2 (Cx26), GJB6 (Cx30), GJB3 (Cx31), GJA1 (Cx43), GJB4 (Cx30.3), and GJB5 (Cx31.1), may cause sensorineural hearing loss and various skin disease phenotypes. The crucial functional importance of each of these connexins in the mentioned ectodermic tissues is reflected by the finding that genetic defects in their genes produce a wide spectrum of genetic disorders comprising sensorineural hearing loss, disorders of cornification of the skin, hair, and nails, and keratitis. Here, we report on different mutations in the connexin genes in individuals with or without hearing loss and different skin disorders illustrating the clinical and genetic heterogeneity of the condition.

Molecular genetics study of deafness in Brazil: 8-year experience.

Hereditary hearing loss is a complex disorder that involves a large number of genes. In developed countries, 1 in 1,000 children is born with deafness severe enough to require special education services, and about 60% of the cases of isolated deafness have a genetic origin. Although more than 100 genes for hearing loss are known currently, only a few are routinely tested in the clinical practice. In this study, we present our findings from the molecular diagnostic screening of the GJB2 and GJB3 genes, del(GJB6-D13S1,830) and del(GJB6-D13S1,854) deletions in the GJB6 gene, Q829X mutation in the otoferlin gene (OTOF) and, the A1,555G and A7,445G mutations in the mitochondrial genome over an 8-year period. Mutations analysis in the previously mentioned genes and mutations was performed on 645 unrelated Brazilian patients with hearing loss who fell into two different testing groups. Different mutations in the GJB2 gene were responsible for most of cases studied, but deletions in the GJB6 gene as well as mitochondrial mutations were also found. While most cases of hearing loss in this country are due to environmental factors, the genetic etiology of deafness will increasingly be determined as more genetic tests become available.

Molecular study in Brazilian cochlear implant recipients.

The most common form of non-syndromic autosomal recessive deafness (NSRD) is caused by mutations in the GJB2 gene. Recently, a deletion truncating the GJB6 gene, called del(GJB6-D13S1,830) has also been described normally accompanying mutations in another allele of the GJB2 gene. Among all the mutations described to date, 35delG in the GJB2 gene is the most common. Preliminary data suggest that pathologic changes due to GJB2 mutations do not affect the spiral ganglion cells, which are the site of stimulation of the cochlear implant. Besides, the survival of the spiral ganglion cells is believed to be an important determinant of the outcome after surgery. Therefore, we have studied 49 non-syndromic deaf patients with unknown etiologies in order to determine the prevalence of GJB2 and GJB6 gene mutations in patients undergoing cochlear implantation surgery. Also, the molecular studies were performed using polymerase chain reaction amplification and direct sequencing. As a result, we found 19 individuals with GJB2 mutation including one new mutation (K168R), one patient homozygous for the del(GJB6-D13S1,830). These results establish that genetic screening can provide an etiologic diagnosis, and may help with prognosis after cochlear implantation, as has been hypothesized in previous studies.