Raul Hernandes Bortolin

Protection against T1DM-Induced Bone Loss by Zinc Supplementation: Biomechanical, Histomorphometric, and Molecular Analyses in STZ-Induced Diabetic Rats

Several studies have established an association between diabetes and alterations in bone metabolism; however, the underlying mechanism is not well established. Although zinc is recognized as a potential preventive agent against diabetes-induced bone loss, there is no evidence demonstrating its effect in chronic diabetic conditions. This study evaluated the effects of zinc supplementation in a chronic (90 days) type 1 diabetes-induced bone-loss model. Male Wistar rats were distributed in three groups: control, type 1 diabetes mellitus (T1DM), and T1DM plus zinc supplementation (T1DMS). Serum biochemical analysis; tibia histomorphometric, biomechanical, and collagen-content analyses; and femur mRNA expression were evaluated. Relative to T1DM, the zinc-supplemented group showed increased histomorphometric parameters such as TbWi and BAr and decreased TbSp, increased biomechanical parameters (maximum load, stiffness, ultimate strain, and Young’s modulus), and increased type I collagen content. Interestingly, similar values for these parameters were observed between the T1DMS and control groups. These results demonstrate the protective effect of zinc on the maintenance of bone strength and flexibility. In addition, downregulation of OPG, COL1A, and MMP-9 genes was observed in T1DMS, and the anabolic effects of zinc were evidenced by increased OC expression and serum ALP activity, both related to osteoblastogenesis, demonstrating a positive effect on bone formation. In contrast, T1DM showed excessive bone loss, observed through reduced histomorphometric and biomechanical parameters, characterizing diabetes-associated bone loss. The bone loss was also observed through upregulation of OPG, COL1A, and MMP-9 genes. In conclusion, zinc showed a positive effect on the maintenance of bone architecture and biomechanical parameters. Indeed, OC upregulation and control of expression of OPG, COL1A, and MMP-9 mRNAs, even in chronic hyperglycemia, support an anabolic and protective effect of zinc under chronic diabetic conditions. Furthermore, these results indicate that zinc supplementation could act as a complementary therapy in chronic T1DM.

Low bone mineral density in patients with type 1 diabetes: association with reduced expression of IGF1, IGF1R and TGF B 1 in peripheral blood mononuclear cells.

The negative effects of type 1 diabetes (T1D) on growth factors of bone metabolism lead to a reduction in bone mineral density. This study aimed to evaluate the association between bone mineral density and insulin‐like growth factor 1 (IGF1), insulin‐like growth factor 1 receptor (IGF1R) and transforming growth factor beta 1 (TGFB1) expressions in children and adolescents with T1D. Moreover, the influences of age at diagnosis, time since diagnosis, glycaemic control and albuminuria on bone mineral density were investigated.

Anabolic Effect of Insulin Therapy on the Bone: Osteoprotegerin and Osteocalcin Up-Regulation in Streptozotocin-Induced Diabetic Rats.

Type 1 diabetes mellitus (T1DM) is associated with several skeletal alterations, particularly in conditions of poor glycaemic control. Insulin therapy is the major conservative treatment for T1DM; however, the effects of this hormone on bone markers of T1DM rats are limited, and the regulatory mechanisms remain elusive. Therefore, the evaluation of molecular and non‐molecular parameters in a chronic animal model of T1DM‐induced bone loss, treated with and without insulin, may help in elucidating the insulin mechanisms. Male Wistar rats were assigned into three groups: control, T1DM (T1DM rats induced with streptozotocin [STZ] at 40 mg/kg intravenously) and T1DM plus insulin therapy (T1DMI). After 8 weeks, we evaluated the serum biochemical, tibia histomorphometric and biomechanical parameters, as well as the gene expression of the receptor activator of nuclear factor kappa‐B ligand (RANKL), osteoprotegerin (OPG) and osteocalcin (OC) of femur mRNA. Compared with T1DM, the T1DMI group showed less bone loss, which was revealed by the increased trabecular width (TbWi, p < 0.001) and trabecular bone area (BAr, p < 0.01), reduced trabecular separation (TbSp, p < 0.01) and increased Youngs modulus (p < 0.05). Moreover, molecular analyses indicated that the expression of OPG and OC was up‐regulated (p < 0.001 and p < 0.05, respectively). In summary, the up‐regulation of OPG and OC in the T1DMI group supports an anabolic effect of insulin, which was demonstrated by the maintenance of bone architecture and flexibility. These results suggest that insulin therapy may prevent T1DM‐induced bone loss via the effects on the bone formation.

Integrated analysis of miRNA and mRNA gene expression microarrays: Influence on platelet reactivity, clopidogrel response and drug-induced toxicity.

Genetic and epigenetic variability may influence the efficacy and safety of antiplatelet therapies, including clopidogrel. Therefore, the miRNA-mRNA interactions and drug toxicity were investigated in silico using available microarray data. Expressions profiles of platelet miRNA (GSE59488) from acute coronary syndrome and mRNA in peripheral blood cells (GSE32226) from coronary artery disease patients were used to miRNA-target mRNA integrated analysis by Ingenuity Pathways Analysis 6 software (IPA). Results showed that ST13 mRNA is regulated by hsa-miR-107 (miR-103-3p); BTNL3 and CFD mRNAs are regulated by hsa-miR-4701-3p (miR-1262); SLC7A8 is regulated by hsa-miR-145-5p (miR-145-5p); and SENP5 mRNA is regulated by hsa-miR-15b-5p (miR-16-5p) and hsa-miR-26a-5p (miR-26a-5p). Drug toxicity IPA tool showed that these miRNAs/mRNAs are associated with clopidogrel-related liver and renal injury. In conclusion, these results demonstrate that differential expression of miRNAs in platelets and interactions with their target mRNAs are associated with variability in platelet reactivity, clopidogrel response and drug-induced toxicity.

Zinc supplementation reduces RANKL/OPG ratio and prevents bone architecture alterations in ovariectomized and type 1 diabetic rats

Type 1 diabetes mellitus (T1DM) and estrogen deficiency are associated with several alterations in bone turnover. Zinc (Zn) is required for growth, development, and overall health. Zinc has been used in complementary therapy against bone loss in several diseases. We hypothesized that Zn supplementation represents a potential therapy against severe bone loss induced by the combined effect of estrogen deficiency and T1DM. We evaluated the protective effect of Zn against bone alterations in a chronic model of these disorders. Female Wistar rats were ramdomized into 3 groups (5 rats each): control, OVX/T1DM (ovariectomized rats with streptozotocin-induced T1DM), and OVX/T1DM+Zn (OVX/T1DM plus daily Zn supplementation). Serum biochemical, bone histomorphometric, and molecular analyses were performed. Histomorphometric parameters were similar between the control and OVX/T1DM+Zn groups, suggesting that Zn prevents bone architecture alterations. In contrast, the OVX/T1DM group showed significantly lower trabecular width and bone area as well as greater trabecular separation than the control. The OVX/T1DM and OVX/T1DM+Zn groups had significantly higher serum alkaline phosphatase activity than the control. The supplemented group had higher levels of serum-ionized calcium and phosphorus than the nonsupplemented group. The RANKL/OPG ratio was similar between the control and OVX/T1DM+Zn groups, whereas it was higher in the OVX/T1DM group. In conclusion, Zn supplementation prevents bone alteration in chronic OVX/T1DM rats, as demonstrated by the reduced RANKL/OPG ratio and preservation of bone architecture. The findings may represent a novel therapeutic approach to preventing OVX/T1DM-induced bone alterations.

Association of insertion-deletions polymorphisms with colorectal cancer risk and clinical features

AIM To investigate the association between 16 insertion-deletions (INDEL) polymorphisms, colorectal cancer (CRC) risk and clinical features in an admixed population. METHODS One hundred and forty patients with CRC and 140 cancer-free subjects were examined. Genomic DNA was extracted from peripheral blood samples. Polymorphisms and genomic ancestry distribution were assayed by Multiplex-PCR reaction, separated by capillary electrophoresis on the ABI 3130 Genetic Analyzer instrument and analyzed on GeneMapper ID v3.2. Clinicopathological data were obtained by consulting the patients’ clinical charts, intra-operative documentation, and pathology scoring. RESULTS Logistic regression analysis showed that polymorphism variations in IL4 gene was associated with increased CRC risk, while TYMS and UCP2 genes were associated with decreased risk. Reference to anatomical localization of tumor Del allele of NFKB1 and CASP8 were associated with more colon related incidents than rectosigmoid. In relation to the INDEL association with tumor node metastasis (TNM) stage risk, the Ins alleles of ACE, HLAG and TP53 (6 bp INDEL) were associated with higher TNM stage. Furthermore, regarding INDEL association with relapse risk, the Ins alleles of ACE, HLAG, and UGT1A1 were associated with early relapse risk, as well as the Del allele of TYMS. Regarding INDEL association with death risk before 10 years, the Ins allele of SGSM3 and UGT1A1 were associated with death risk. CONCLUSION The INDEL variations in ACE, UCP2, TYMS, IL4, NFKB1, CASP8, TP53, HLAG, UGT1A1, and SGSM3 were associated with CRC risk and clinical features in an admixed population. These data suggest that this cancer panel might be useful as a complementary tool for better clinical management, and more studies need to be conducted to confirm these findings.

Albuminuria and low bone mineral density in paediatric patients with type 1 diabetes: Type 1 diabetes: Albuminuria/Bone loss

To evaluate glycaemic control and its influence on albuminuria and bone mineral density (BMD) in children and adolescents with type 1 diabetes (T1D).

Application of high-resolution array platform for genome-wide copy number variation analysis in patients with nonsyndromic cleft lip and palate

Although more than 14 loci may be involved in the development of nonsyndromic cleft lip and palate (NSCLP), the etiology has not been fully elucidated due to genetic and environmental risk factor interactions. Despite advances in identifying genes associated with the NSCLP development using traditional genetic mapping strategies of candidate genes, genome‐wide studies, and epidemiologic and linkage analysis, microarray techniques have become important complementary tools in the search for potential causative oral clefts genes in genetic studies. Microarray hybridization enables scanning of the whole genome and detecting copy number variants (CNVs). Although common benign CNVs are often smaller, with sizes smaller than 20 kb, here we reveal small exonic CNVs based on the importance of the encompassed genes in cleft lip and palate phenotype.

Relationship between circulating VCAM-1, ICAM-1, E-selectin and MMP9 and the extent of coronary lesions

OBJECTIVES: Inflammatory molecules play a role in the development of atherosclerosis, which is the primary origin of cardiovascular disorders. However, to the best of our knowledge, no study has attempted to investigate the relationship between these circulating molecules and the prediction of cardiovascular risk. The present study aimed to investigate the relationships of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 serum concentrations with the extent of coronary lesions. METHODS: Seventy-four individuals who were undergoing coronary angiography for the first time for diagnostic purposes were enrolled in this study. The extent of the coronary lesion was assessed using the Friesinger Index, and subjects were classified into four groups: no lesions, minor lesions, intermediate lesions and major lesions. Serum biochemical parameters and serum concentrations of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, E-selectin and matrix metalloproteinase 9 were analyzed. RESULTS: The vascular cell adhesion molecule-1 concentration was higher than 876 ng/mL in individuals with intermediate and major lesions (p<0.001 and p=0.020, respectively). Moreover, logistic regression analysis showed that these patients had an increased risk of having an intermediate lesion (p=0.007). Interestingly, all individuals with major lesions had vascular cell adhesion molecule-1 concentrations higher than 876 ng/mL. No association was found between the concentrations of the other proteins and the Friesinger Index. CONCLUSIONS: Serum vascular cell adhesion molecule-1 may be associated with the extent of coronary lesions. Moreover, it may represent an alternative to improve the cardiovascular risk classification in patients without acute coronary syndrome.

Risk factors and comorbidities in Brazilian patients with orofacial clefts

Considering that environmental risk factors substantially contribute to the etiology of orofacial clefts and that knowledge about the characteristics and comorbidities associated with oral clefts is fundamental to promoting better quality of life, this study aimed to describe the risk factors, main characteristics, and comorbidities of a group of patients with cleft lip and/or cleft palate (CL/P) from Rio Grande do Norte (RN), Brazil. Data were obtained from 173 patients with CL/P using a form from the Brazilian database on Orofacial Clefts. Most patients were male with cleft lip and palate and had a normal size and weight at birth; presented few neonatal intercurrent events; and had anemia and respiratory and cardiovascular diseases as main associated comorbidities. They also required timely surgical rehabilitation and multidisciplinary care to stimulate their neuropsychomotor development. In addition, a high frequency of familial recurrence and of parental consanguinity was evidenced in the studied population, especially for the cleft lip and cleft palate type. Other relevant findings were the considerable maternal exposure to alcohol, infections, smoking, and hypertension, as well as low supplementation with vitamins and minerals and deliberate consumption of analgesics, antibiotics, and antihypertensives during pregnancy. Characterization of the CL/P patient profile is essential for the planning of health services and integration among the health professionals involved in the diagnosis and treatment of these malformations. Our results reinforce the need for additional research to confirm the association between environmental factors and the development of orofacial clefts.