Disruptive 3D in vitro models for respiratory disease investigation: A state-of-the-art approach focused on SARS-CoV-2 infection.

COVID-19, along with most respiratory diseases in the medical field, demonstrates significant ability to take its toll on global population. There is a particular difficulty in studying these conditions, which stems especially from the short supply of in vitro models for detailed investigation, the specific therapeutic knowledge required for disease scrutinization and the occasional need of BSL-3 [Biosafety Level 3] laboratories for research. Based on this, the process of drug development is hampered to a great extent. In the scenario of COVID-19, this difficulty is even more substantial on account of the current undefinition regarding the exact role of the ACE2 [Angiotensin-converting enzyme 2] receptor upon SARS-CoV-2 kinetics in human cells and the great level of demand in the investigation process of ACE2, which usually requires the laborious and ethically complicated usage of transgenic animal models overexpressing the receptor. Moreover, the rapid progression of the aforementioned diseases, especially COVID-19, poses a crucial necessity for adequate therapeutic solutions emergence. In this context, the work herein presented introduces a groundbreaking set of 3D models, namely spheroids and MatriWell cell culture inserts, whose remarkable ability to mimic the in vivo environment makes them highly suitable for respiratory diseases investigation, particularly SARS-CoV-2 infection. Using MatriWells, we developed an innovative platform for COVID-19 research: a pulmonary air-liquid interface [ALI] associated with endothelial (HUVEC) cells. Infection studies revealed that pulmonary (BEAS-2B) cells in the ALI reached peak viral load at 24h and endothelial cells, at 48h, demonstrating lung viral replication and subsequent hematogenous dissemination, which provides us with a unique and realistic framework for studying COVID-19. Simultaneously, the spheroids were used to address the understudied ACE2 receptor, aiming at a pronounced process of COVID-19 investigation. ACE2 expression not only increased spheroid diameter by 20% (p<0.001) and volume by 60% (p≤0.0001) but also led to a remarkable 640-fold increase in intracellular viral load (p≤0.01). The previously mentioned finding supports ACE2 as a potential target for COVID-19 treatment. Lastly, we observed a higher viral load in the MatriWells compared to spheroids (150-fold, p<0.0001), suggesting the MatriWells as a more appropriate approach for COVID-19 investigation. By establishing an advanced method for respiratory tract conditions research, this work paves the way toward an efficacious process of drug development, contributing to a change in the course of respiratory diseases such as COVID-19.

Bioactive decellularized cardiac extracellular matrix-based hydrogel as a sustained-release platform for human adipose tissue-derived stromal cell-secreted factors.

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-1ß, 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.

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 tubulointerstitialinflammation associated with the increase of fibrosis through the excess deposition of extracellular matrix (ECM) whichleads to decrease glomerular filtration. Many different underlying renal diseases can affect the kidneys of dogs such ascongenital or acquired in origin. Therefore, the main objective of this transversal study was to evaluate the epidemiologythrough clinical and laboratory evaluation of 225 client-owned dogs with CKD.Materials, Methods & Results: Complete blood count (CBC), urinalysis, and biochemical profile were retrospectivelyselected and evaluated from 225 client-owned dogs with CKD of both sexes, different ages, and breeds from the patientpopulation of the Nephrology and Urology Small Animal Service of the Teaching Hospital of the School of VeterinaryMedicine 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 toKruskal-Wallis non-parametric test complemented with Dunns multiple comparisons test, and analysis of variance for themodel with a factor complemented with the test of multiple comparisons of Tukey. In this retrospective study, we observedthat most dogs in all groups were elderly (≥ 9 years old). CBC demonstrated lower RBCs (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 notdemonstrate 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...(AU)

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 tubulointerstitialinflammation associated with the increase of fibrosis through the excess deposition of extracellular matrix (ECM) whichleads to decrease glomerular filtration. Many different underlying renal diseases can affect the kidneys of dogs such ascongenital or acquired in origin. Therefore, the main objective of this transversal study was to evaluate the epidemiologythrough clinical and laboratory evaluation of 225 client-owned dogs with CKD.Materials, Methods & Results: Complete blood count (CBC), urinalysis, and biochemical profile were retrospectivelyselected and evaluated from 225 client-owned dogs with CKD of both sexes, different ages, and breeds from the patientpopulation of the Nephrology and Urology Small Animal Service of the Teaching Hospital of the School of VeterinaryMedicine 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 toKruskal-Wallis non-parametric test complemented with Dunn’s multiple comparisons test, and analysis of variance for themodel with a factor complemented with the test of multiple comparisons of Tukey. In this retrospective study, we observedthat 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 notdemonstrate 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...

Adipose tissue-derived stromal cells' conditioned medium modulates endothelial-mesenchymal transition induced by IL-1ß/TGF-ß2 but does not restore endothelial function.

OBJECTIVES: Endothelial cells undergo TGF-ß-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-1ß and TGF-ß2 to induce EndMT, and the influence of ASC-CMed was assessed. As controls, non-treated HUVEC or HUVEC treated only with IL-1ß 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-1ß/TGF-ß2 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-1ß/TGF-ß2-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.

Fibroblast growth factor-2, but not the adipose tissue-derived stromal cells secretome, inhibits TGF-ß1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

Transforming growth factor-ß1 (TGF-ß1) 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-ß1 signaling. We hypothesized that TGF-ß1-induced cardiac fibroblast to myofibroblast differentiation is abrogated by FGF-2 and ASC conditioned medium (ASC-CMed). Our experiments demonstrated that TGF-ß1 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 < 0.0001; ACTA2, p = 0.0056) and protein (αSMA, p = 0.0338) expression of mesenchymal markers and extracellular matrix components gene expression (COL1A1, p < 0.0001; COL3A1, p = 0.0029). ASC-CMed did not block myofibroblast differentiation. The treatment with FGF-2 increased matrix metalloproteinases gene expression (MMP1, p < 0.0001; 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-ß1-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.