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Transforming growth factor-β1 (TGF-β1) is a potent inducer of fibroblast to myofibroblast differentiation and contributes to the pro-fibrotic microenvironment during cardiac remodeling. Fibroblast growth factor-2 (FGF-2) is a growth factor secreted by adipose tissue-derived stromal cells (ASC) which can antagonize TGF-β1 signaling. We hypothesized that TGF-β1-induced cardiac fibroblast to myofibroblast differentiation is abrogated by FGF-2 and ASC conditioned medium (ASC-CMed). Our experiments demonstrated that TGF-β1 treatment-induced cardiac fibroblast differentiation into myofibroblasts, as evidenced by the formation of contractile stress fibers rich in αSMA. FGF-2 blocked the differentiation, as evidenced by the reduction in gene (TAGLN, p ACTA2, p = 0.0056) and protein (αSMA, p = 0.0338) expression of mesenchymal markers and extracellular matrix components gene expression (COL1A1, p COL3A1, p = 0.0029). ASC-CMed did not block myofibroblast differentiation. The treatment with FGF-2 increased matrix metalloproteinases gene expression (MMP1, p MMP14, p = 0.0027) and decreased the expression of tissue inhibitor of metalloproteinase gene TIMP2 (p = 0.0023). ASC-CMed did not influence these genes. The proliferation of TGF-β1-induced human cardiac fibroblasts was restored by both FGF-2 (p = 0.0002) and ASC-CMed (p = 0.0121). The present study supports the anti-fibrotic effects of FGF-2 through the blockage of cardiac fibroblast differentiation into myofibroblasts. ASC-CMed, however, did not replicate the anti-fibrotic effects of FGF-2 in vitro.

The administration of trophic factors (TFs) released by mesenchymal stromal cells (MSCs) as therapy for cardiovascular diseases requires a delivery vehicle capable of binding and releasing the TF in a sustained manner. We hypothesized that hydrogels derived from cardiac decellularized extracellular matrix (cardiac dECM) bind MSC secretome-derived TF and release these in a sustained fashion. Pig-derived ventricular tissue was decellularized, milled to powder, digested, and assembled as a hydrogel upon warming at 37 °C. The conditioned medium (CMed) of adipose tissue-derived stromal cells (ASC) was collected, concentrated, and incorporated into the hydrogel at 1×, 10×, and 100× the original concentration. The release of 11 ASC-secreted factors (angiopoietin-1, angiopoietin-2, fibroblast growth factor-1, hepatocyte growth factor, platelet-derived growth factor-AA, vascular endothelial growth factor, interleukin-1β, interleukin-6, interleukin-8, CCL2, and matrix metalloproteinase-1) from hydrogels was immune assessed. Bioactivity was determined by endothelial cell proliferation, function, and assessment of endothelial mesenchymal transition. We showed that dECM hydrogels could be loaded with human ASC-secreted TFs, which are released in a sustained manner for several days subsequently. Different trophic factors had different release kinetics, which correlates with the initial concentration of CMed in the hydrogel. We observed that the more concentrated was the hydrogel, the more inflammation-related cytokines, and the less pro-regenerative TFs were released. Finally, we showed that the factors secreted by the hydrogel are biologically active as these influence cell behavior. The use of dECM hydrogels as a platform to bind and release paracrine factors secreted by (mesenchymal) cells is a potential alternative in the context of cardiovascular regeneration.

It is known that guanosine derivatives (G) self-assemble in water forming long, flexible, and interacting aggregates (the so-called G-quadruplexes): by modulating the quadruplex charges, e.g. simply using a mixture of guanosine 5'-monophosphate (GMP) and guanosine (Gua), multi-responsive, self-healing hydrogels can be obtained. In this paper, the potential application of G-hydrogels as drug delivery systems has been assessed. Hydrogels were prepared at different Gua:GMP molar ratios. The photosensitizer Methylene Blue and the pro-apoptotic protein cytochrome C were used as cargo molecules. Small angle x-ray scattering and atomic force microscopy experiments confirmed the presence of G-quadruplexes disposed in swollen matrices with different mesh-sizes. Rheology measurements showed that the Gua:GMP molar ratio leads to specific drug release mechanisms, as the gel strength is finely tuned by electrostatic repulsion and van der Waals attraction between G-quadruplexes. Noteworthy, the gel cohesion and the drug release were pH responsive. Swelling, self-healing and cell viability features were also investigated: the results qualify the Gua:GMP hydrogel as an excellent biomaterial that can entrap and deliver key biomolecules in a sustained and responsive release manner.

Made available in DSpace on 2020-12-10T20:00:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-29 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: Chronic kidney disease (CKD) affects both dogs and cats, mainly elderly animals, due to tubulointerstitial inflammation associated with the increase of fibrosis through the excess deposition of extracellular matrix (ECM) which leads to decrease glomerular filtration. Many different underlying renal diseases can affect the kidneys of dogs such as congenital or acquired in origin. Therefore, the main objective of this transversal study was to evaluate the epidemiology through clinical and laboratory evaluation of 225 client-owned dogs with CKD. Materials, Methods & Results: Complete blood count (CBC), urinalysis, and biochemical profile were retrospectively selected and evaluated from 225 client-owned dogs with CKD of both sexes, different ages, and breeds from the patient population of the Nephrology and Urology Small Animal Service of the Teaching Hospital of the School of Veterinary Medicine and Animal Science - Sao Paulo State University from 2011 to 2017. All dogs were divided in groups according to the International Renal Interest Society (IRIS) CKD grading and statistical analysis was performed according to Kruskal-Wallis non-parametric test complemented with Dunn's multiple comparisons test, and analysis of variance for the model with a factor complemented with the test of multiple comparisons of Tukey. In this retrospective study, we observed that most dogs in all groups were elderly (>= 9 years old). CBC demonstrated lower RBC's (P < 0.005), hemoglobin (P < 0.001), hematocrit (Ht%) [P < 0.001] at the highest stage of the disease. However, urinary specific gravity (USG) did not demonstrate significant differences between the disease stages, but urinary protein: creatinine ratio (UPC) was statistically different (P < 0.01) between IRIS-CKD stages 1 and 4. Furthermore, serum phosphate concentrations demonstrated significantly higher levels in dogs at IRI-CKD stage 4 compared with IRIS-CKD stage 3 (P < 0.001). Discussion: The analysis of 225 dogs with CKD showed that 130 animals were elderly, older than 9 years, and according to previous studies, 15% of dogs over 10 years of age are diagnosed of CKD, presenting significant morbidity and mortality. Laboratory findings such as the presence of non-regenerative anemia is expected in dogs with CKD. In our study, the degree of anemia corresponded with the stage of the disease, similarly to serum creatinine concentrations. Another important laboratory finding in diagnosing CKD is the early detection of the kidney's abilities in concentrating its tubular filtrate. In this retrospective study, isosthenuria was not significant due to all dogs presented CKD as criteria of inclusion, especially those without azotemia, although, proteinuria was reported in 90% of the population investigated. Electrolyte imbalances are also expected in CKD. However, despite serum sodium, potassium, and total calcium did not demonstrate significant results, serum phosphate had its significance between IRIS-CKD stage 3 and stage 4. Hence, despite the age factor of most dogs in all groups, with the association of laboratory results such as serum creatinine, serum phosphorus, ionized calcium, erythrogram, isosthenuria, SBP, and the degree of proteinuria, it was possible to perform early diagnosis of CKD even in dogs with IRIS-CKD Stage 1 in a stable and hydrated patient. With the proper diagnostic, staging and substaging according to IRIS guidelines, these parameters can be monitored, predicting longevity and good quality of life, or progression of the disease with a more reserved prognosis. Sao Paulo State Univ, UNESP, Vet Clin Dept, Botucatu, SP, Brazil Sao Paulo State Univ, UNESP, Dept Anim Reprod & Radiol, Sch Vet Med & Anim Sci, Botucatu, SP, Brazil Sao Paulo State Univ, UNESP, Dept Biostat, Sch Biosci, Botucatu, SP, Brazil Univ Sao Paulo, Dept Thorac & Cardiovasc Surg, Sao Paulo, SP, Brazil Sao Paulo State Univ, UNESP, Vet Clin Dept, Botucatu, SP, Brazil Sao Paulo State Univ, UNESP, Dept Anim Reprod & Radiol, Sch Vet Med & Anim Sci, Botucatu, SP, Brazil Sao Paulo State Univ, UNESP, Dept Biostat, Sch Biosci, Botucatu, SP, Brazil FAPESP: 2012/16752-9

OBJECTIVES: Endothelial cells undergo TGF-β-driven endothelial-mesenchymal transition (EndMT), representing up to 25% of cardiac myofibroblasts in ischaemic hearts. Previous research showed that conditioned medium of adipose tissue-derived stromal cells (ASC-CMed) blocks the activation of fibroblasts into fibrotic myofibroblasts. We tested the hypothesis that ASC-CMed abrogates EndMT and prevents the formation of adverse myofibroblasts.MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were treated with IL-1β and TGF-β2 to induce EndMT, and the influence of ASC-CMed was assessed. As controls, non-treated HUVEC or HUVEC treated only with IL-1β in the absence or presence of ASC-CMed were used. Gene expression of inflammatory, endothelial, mesenchymal and extracellular matrix markers, transcription factors and cell receptors was analysed by RT-qPCR. The protein expression of endothelial and mesenchymal markers was evaluated by immunofluorescence microscopy and immunoblotting. Endothelial cell function was measured by sprouting assay.RESULTS: IL-1β/TGF-β2 treatment induced EndMT, as evidenced by the change in HUVEC morphology and an increase in mesenchymal markers. ASC-CMed blocked the EndMT-related fibrotic processes, as observed by reduced expression of mesenchymal markers TAGLN (P = 0.0008) and CNN1 (P = 0.0573), as well as SM22α (P = 0.0501). The angiogenesis potential was impaired in HUVEC undergoing EndMT and could not be restored by ASC-CMed.CONCLUSIONS: We demonstrated that ASC-CMed reduces IL-1β/TGF-β2-induced EndMT as observed by the loss of mesenchymal markers. The present study supports the anti-fibrotic effects of ASC-CMed through the modulation of the EndMT process.

Animal experimentation requires a solid and rational moral foundation. Objective and emphatic decision-making and protocol evaluation by researchers and ethics committees remain a difficult and sensitive matter. This article presents three perspectives that facilitate a consideration of the minimally acceptable standard for animal experiments, in particular, in tissue engineering (TE) and regenerative medicine. First, we review the boundaries provided by law and public opinion in America and Europe. Second, we review contemporary moral theory to introduce the Neo-Rawlsian contractarian theory to objectively evaluate the ethics of animal experiments. Third, we introduce the importance of available reduction, replacement, and refinement strategies, which should be accounted for in moral decision-making and protocol evaluation of animal experiments. The three perspectives are integrated into an algorithmic and graphic harm-benefit analysis tool based on the most relevant aspects of animal models in TE. We conclude with a consideration of future avenues to improve animal experiments.

Introduction. Progenitor cells cultured on biomaterials with optimal physical-topographical properties respond with alignment and differentiation. Stromal cells from connective tissue can adversely differentiate to profibrotic myofibroblasts or favorably to smooth muscle cells (SMC). We hypothesized that myogenic differentiation of adipose tissue-derived stromal cells (ASC) depends on gradient directional topographic features. Methods. Polydimethylsiloxane (PDMS) samples with nanometer and micrometer directional topography gradients (wavelength w=464-10, 990 nm; amplitude a=49-3, 425 nm) were fabricated. ASC were cultured on patterned PDMS and stimulated with TGF-β1 to induce myogenic differentiation. Cellular alignment and adhesion were assessed by immunofluorescence microscopy after 24 h. After seven days, myogenic differentiation was examined by immunofluorescence microscopy, gene expression, and immunoblotting. Results. Cell alignment occurred on topographies larger than w=1758 nm/a=630 nm. The number and total area of focal adhesions per cell were reduced on topographies from w=562 nm/a=96 nm to w=3919 nm/a=1430 nm. Focal adhesion alignment was increased on topographies larger than w=731 nm/a=146 nm. Less myogenic differentiation of ASC occurred on topographies smaller than w=784 nm/a=209 nm. Conclusion. ASC adherence, alignment, and differentiation are directed by topographical cues. Our evidence highlights a minimal topographic environment required to facilitate the development of aligned and differentiated cell layers from ASC. These data suggest that nanotopography may be a novel tool for inhibiting fibrosis.