Adipose tissue-derived stromal cells' conditioned medium modulates endothelial-mesenchymal transition induced by IL-1β/TGF-β2 but does not restore endothelial function

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

Molecular and Biomechanical Clues From Cardiac Tissue Decellularized Extracellular Matrix Drive Stromal Cell Plasticity

Decellularized-organ-derived extracellular matrix (dECM) has been used for many years in tissue engineering and regenerative medicine. The manufacturing of hydrogels from dECM allows to make use of the pro-regenerative properties of the ECM and, simultaneously, to shape the material in any necessary way. The objective of the present project was to investigate differences between cardiovascular tissues (left ventricle, mitral valve, and aorta) with respect to generating dECM hydrogels and their interaction with cells in 2D and 3D. The left ventricle, mitral valve, and aorta of porcine hearts were decellularized using a series of detergent treatments (SDS, Triton-X 100 and deoxycholate). Mass spectrometry-based proteomics yielded the ECM proteins composition of the dECM. The dECM was digested with pepsin and resuspended in PBS (pH 7.4). Upon warming to 37°C, the suspension turns into a gel. Hydrogel stiffness was determined for samples with a dECM concentration of 20 mg/mL. Adipose tissue-derived stromal cells (ASC) and a combination of ASC with human pulmonary microvascular endothelial cells (HPMVEC) were cultured, respectively, on and in hydrogels to analyze cellular plasticity in 2D and vascular network formation in 3D. Differentiation of ASC was induced with 10 ng/mL of TGF-β1 and SM22α used as differentiation marker. 3D vascular network formation was evaluated with confocal microscopy after immunofluorescent staining of PECAM-1. In dECM, the most abundant protein was collagen VI for the left ventricle and mitral valve and elastin for the aorta. The stiffness of the hydrogel derived from the aorta (6,998 ± 895 Pa) was significantly higher than those derived from the left ventricle (3,384 ± 698 Pa) and the mitral valve (3,233 ± 323 Pa) (One-way ANOVA, p = 0.0008). Aorta-derived dECM hydrogel drove non-induced (without TGF-β1) differentiation, while hydrogels derived from the left ventricle and mitral valve inhibited TGF-β1-induced differentiation. All hydrogels supported vascular network formation within 7 days of culture, but ventricular dECM hydrogel demonstrated more robust vascular networks, with thicker and longer vascular structures. All the three main cardiovascular tissues, myocardium, valves, and large arteries, could be used to fabricate hydrogels from dECM, and these showed an origin-dependent influence on ASC differentiation and vascular network formation

Development of cellular matrices for the treatment of dilated cardiomyopathy

Embora muito se tenha avançado no desenvolvimento de novas terapias para as cardiomiopatias, ainda não existe uma opção curativa que substitua a necessidade do transplante. A medicina regenerativa vem se consolidando pelo mundo ao trazer novas alternativas para doenças cardíacas crônicas. Embora os cardiomiócitos sejam afetados de maneira importante no processo de degeneração do órgão, sendo os responsáveis pela contração cardíaca, eles não são, na verdade, as células mais abundantes no coração de mamíferos. Esse lugar é ocupado pelos fibroblastos: células alongadas presentes entre as fibras musculares e que ajudam na manutenção do equilíbrio da matriz extracelular. Durante a insuficiência cardíaca, dois processos contribuem para o desbalanço desse equilíbrio e consequente fibrose e remodelamento do coração. O primeiro processo é a diferenciação de fibroblastos em miofibroblastos; o segundo é a transição de células endoteliais em mesenquimais (EndMT). Ambos os processos levam ao aumento de células responsáveis por depositar matriz extracelular e, portanto, à fibrose e remodelamento. Assim, abordagens direcionadas a impedir o progresso desses processos podem ser uma alternativa interessante para o tratamento das cardiomiopatias. Utilizando-se de células-tronco, biomateriais e fatores de crescimento, o objetivo desta tese é a criação de uma nova abordagem terapêutica que vise diminuir ou extinguir o processo de fibrose no tecido cardíaco, a fim de manter preservada a função do órgão. Para isso em nosso primeiro estudo, Fibroblast growth factor-2, but not the adipose tissuederived stromal cells secretome, inhibits TGF-Beta1-induced differentiation of human cardiac fibroblasts into myofibroblasts (Scientific Reports, 2018) fibroblastos cardíacos humanos primários, foram cultivados e sua diferenciação foi estimulada através do uso de TGF-Beta1 in vitro. Como tentativa de prevenir sua transformação em miofibroblastos, foi realizada a coleta do secretoma de células estromais derivadas de tecido adiposo (ASC-CMed) e os fibroblastos foram tratados com tal secretoma. Neste trabalho, os resultados demonstraram que não foi possível inibir a diferenciação dos fibroblastos em miofibroblastos com o uso de ASC-CMed. Em nosso segundo estudo, Adipose tissue-derived stromal cells\' conditioned medium modulates endothelial-mesenchymal transition induced by IL-1Beta/TGF-Beta2 but does not restore endothelial function (Cell Proliferation, 2019), induzimos a diferenciação de células endoteliais através de um ambiente pró-inflamatório e prófibrótico, utilizando IL-1Beta e TGF-Beta1, e buscamos impedir a diferenciação dessas utilizando ASC-CMed. Os resultados mostraram que, embora as células endoteliais não tenham retornado a um fenótipo funcional, nós conseguimos inibir o processo de EndMT. Nosso terceiro estudo, Bioactive decellularized cardiac extracellular matrix-based hydrogel as a sustained-release platform for human adipose tissue-derived stromal cell-secreted factors (submetido), teve como objetivo desenvolver uma plataforma baseada em um hidrogel derivado de matriz extracelular miocárdica para liberação do ASC-CMed, processo que foi testado utilizando em diferentes concentrações de secretoma (1x, 10x e 100x). Nossos resultados demonstraram que esse biomaterial demonstrou ser uma excelente plataforma para a liberação fatores de crescimento de maneira contínua. Finalizando a presente tese, com a utilização desse mesmo hidrogel, no quarto estudo, Intrapericardial injection of hydrogels derived from decellularized cardiac extracellular matrix loaded with mesenchymal stromal cells and their secretome: a novel therapeutic approach to treat cytostatics-induced dilated cardiomyopathy, nós levamos em consideração a resposta imune in vivo, ao realizarmos a aplicação intrapericárdica do hidrogel carreado de ASC e seu secretoma em um modelo crônico de cardiomiopatia dilatada em ratos. Neste estudo foi possível concluir que nossa terapia foi capaz de diminuir a fibrose cardíaca e reduzir o remodelamento, bem como melhorar a função cardíaca e parâmetros hemodinâmicos dos animais. Assim, o conteúdo da presente tese demonstra que a aplicação intrapericárdica de um hidrogel composto de matriz extracelular miocárdica carreada com células estromais derivadas de tecido adiposo e seus fatores de crescimento proporciona um ambiente que predispõe a regeneração cardíaca. Futuros estudos são necessários para melhor elucidar os mecanismos envolvidos nessa melhora de função, bem como o estudo em modelos animais que mais se assemelham aos humanos, objetivando, em um momento futuro, a aplicação dessa terapia em clinical trialsAlthough considerable progress has been made in the development of new therapies for cardiomyopathies, there is still no curative alternative to replace the need for transplantation. Regenerative medicine has been consolidated around the world by bringing new alternatives to chronic heart disease. Although cardiomyocytes are significantly affected in the cardiac degeneration process, being responsible for the muscle contraction, they are not, in fact, the most abundant cells in the heart of mammals. This place is occupied by fibroblasts: elongated cells present between the muscle fibers and which help to maintain the extracellular matrix balance. During heart failure, two processes contribute to the disruption of this balance and the consequent fibrosis and heart remodeling. The first process is the transdifferentiation of cardiac fibroblasts into myofibroblasts; the second is the endothelial-mesenchymal transition (EndMT). Both processes lead to an increase in the number of cells responsible for depositing extracellular matrix and, therefore, fibrosis and remodeling. Thus, approaches aimed at preventing the progress of these processes can be an interesting alternative for the treatment of cardiomyopathies. Using stem cells, biomaterials and growth factors, the objective of this thesis was to create a new therapeutic approach to decrease or extinguish the process of fibrosis in the cardiac tissue, in order to maintain the organ\'s function preserved. For that, in our first study, Fibroblast growth factor-2, but not the adipose tissue-derived stromal cells secretome, inhibits TGF-Beta1-induced differentiation of human cardiac fibroblasts into myofibroblasts (Scientific Reports, 2018), primary human cardiac fibroblasts stimulated TGF-Beta1 in vitro and their transdifferentiation was assessed. In an attempt to prevent its transformation into myofibroblasts, the secretome of adipose tissuederived stromal cells (ASC-CMed) was collected and the fibroblasts were treated with it. In this work, the results demonstrated that it was not possible to inhibit the transdifferentiation of fibroblasts into myofibroblasts with the use of ASC-CMed. In our second study, Adipose tissue-derived stromal cells\' conditioned medium modulates endothelial-mesenchymal transition induced by IL-1Beta / TGF-Beta2 but does not restore endothelial function (Cell Proliferation, 2019), we induced the differentiation of endothelial cells through a pro-inflammatory and pro-fibrotic environment, using IL-1Beta and TGF-Beta1, and we sought to prevent their differentiation using ASC-CMed. The results showed that, although the endothelial cells did not return to a functional phenotype, we were able to inhibit the EndMT process. Our third study, Bioactive decellularized cardiac extracellular matrix-based hydrogel as a sustained-release platform for human adipose tissue-derived stromal cellsecreted factors (submitted), aimed to develop a platform based on a hydrogel derived from myocardial extracellular matrix for the release of ASC-CMed, a process that was tested using different concentrations of secretome (1X, 10X and 100X). Our results demonstrated that this biomaterial proved to be an excellent platform for releasing growth factors in a sustained manner. Concluding the present thesis, using this same hydrogel, in the fourth study, Intrapericardial injection of hydrogels derived from decellularized cardiac extracellular matrix loaded with mesenchymal stromal cells and their secretome: a novel therapeutic approach to treat cytostatics-induced dilated cardiomyopathy, we took into account the immune response in vivo, when carrying out the intrapericardial application of the dECM hydrogel loaded with ASC and their secretome in a rat model of dilated cardiomyopathy. In this study it was possible to conclude that our therapy was able to decrease cardiac fibrosis and reduce remodeling, as well as improving cardiac function and hemodynamic parameters of the treated animals. Thus, the content of the present thesis demonstrates that the intrapericardial application of a myocardial extracellular matrix-derived hydrogel loaded with ASC and their growth factors constitutes a potential approach for cardiac regeneration. Further studies are necessary to better elucidate the mechanisms involved in the process, as well as to investigate the approach in large animal models that better resemble humans, aiming, in the future, the application of this therapy in clinical trial

Adipose tissue–derived stromal cells’ conditioned medium modulates endothelial‐mesenchymal transition induced by IL‐1β/TGF‐β2 but does not restore endothelial function

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

Directional Topography Influences Adipose Mesenchymal Stromal Cell Plasticity: Prospects for Tissue Engineering and Fibrosis

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

A Transversal Study of Biochemical Profile, Urinalysis, UPC, Electrolytes and Blood Pressure in Dogs with Chronic Kidney Disease

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 - São 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