Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis

Introduction: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. Materials and Methods: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. Results: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. Conclusion: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC. .

Avaliação dos glicosaminoglicanos do tecido periuretral de pacientes com e sem prolapso genital / Evaluation of glycosaminoglycans of periurethral tissue in patients with and without pelvic organ prolapse

OBJETIVOS: Caracterizar e quantificar os subtipos de glicosaminoglicanos sulfatados (GAGs) existentes no tecido peri-uretral de pacientes com e sem prolapso genital. METODOS: Foram incluídas 35 pacientes que se submeteram a cirurgia vaginal para correção de distopias genitais e/ou incontinência urinária de esforço ou por outra condição benigna. As pacientes foram avaliadas por anamnese padronizada, exame físico e urodinâmico e agrupadas segundo a existência do prolapso genital. Durante o procedimento cirúrgico, amostras de aproximadamente 1,0 x 1,0 cm do tecido periuretral foram retiradas para avaliação. Os GAGs foram extraídos do tecido por proteólise e precipitação por ácido tricloroacético e caracterizados por eletroforese em gel de agarose. A quantificação foi feita por meio de densitometria a 525 nm do gel corado com azul de toluidina. Compararam-se os dados pela análise de variância (ANOVA). RESULTADOS: Nos grupos estudados, houve maior predomínio de dermatam sulfato (DS), em torno de 85 por cento do total de GAGs, seguido do condroitim sulfato (CS) e do heparam sulfato (HS). Observou-se aumento significativo dos GAGs totais, do DS e do HS em mulheres com prolapso genital. Não se observou diferença significante com relação ao CS. CONCLUSÃO: Este estudo demonstrou diferenças na matriz extracelular do tecido periuretral com aumento de GAGs totais, DS e HS nas mulheres com prolapso genital.

OBJECTIVE: To characterize and quantify periurethral tissue sulphated glycosaminoglycans (GAGs) in women with and without pelvic organ prolapse. STUDY DESIGN: Periurethral tissue was obtained from 35 women who underwent surgery for pelvic organ prolapse, for stress urinary incontinence, or for other gynecological benign conditions. Patients were submitted to a clinical history, physical and urodynamic examination and were divided in two groups according to genital prolapse. The standard biopsy with 1.0 x 1.0 cm was taken from periurethral tissue during surgery and assessed by biochemical methods. The GAGs were obtained by proteolysis and precipitated by trichloroacetic acid. The relative concentration of sulfated GAGs was determined by densitometry of toluidine blue stained gel using a spectrophotometer with a 525 nm wavelength. Data were compared using analysis of variance (ANOVA). RESULTS: In the two groups dermatan sulphate (DS) was the predominant glycosaminoglycan (85 percent), followed by chondroitin sulphate (CS) and heparan sulphate (HS). Women with pelvic organ prolapse had significantly more total GAGs, DS and HS. Differences in CS were not observed. CONCLUSIONS: This study showed altered biochemical characteristics in the extracellular matrix of periurethral tissue and also accumulation of GAGs, DS and CS, in women with pelvic organ prolapse.