Systematic review of circulating MICRORNAS as biomarkers of cervical carcinogenesis.

BACKGROUND: Cervical cancer is a preventable disease, but it is a major public health problem despite having a good prognosis when diagnosed early. Although the Pap smear has led to huge drops in rates of cervical cancer and death from the disease, it has some limitations, making new approaches necessary for early diagnosis and biomarkers discovery. MiRNAs have been considered a new class of non-invasive biomarkers and may have great clinical value for screening early-stage cervical intraepithelial neoplasia. Well-designed studies have emerged as a necessary strategy for the identification of miRNAs that could be used safely and reliably for a differential diagnosis. This review aims to provide an up-to-date perspective on the assessment of circulating miRNA expression from precursor lesions to cervical cancer, identifying circulating miRNAs or specific miRNA signatures that can be used as potential biomarkers of different stages of cervical carcinogenesis. METHODS: A systematic review was performed and searches were conducted in the PubMed, LILACS, and Scopus electronic databases. RESULTS: Most studies involved Chinese ethnic women and searched for circulating miRNAs in serum samples. Thirty three microRNAs were evaluated in the eligible studies and 17 (miR-196a, miR-16-2, miR-497, miR-1290, miR-425-5p, hsa-miR- 92a, miR-1266, miR-9, miR-192, miR-205, miR-21, miR-152, miR-15b, miR-34a, miR-218, miR-199a-5p and miR-155-5p) showed up-regulation in women with precursor lesion and cervical cancer and 16 microRNAs showed decreased expression in these same groups of women compared to healthy controls (miR-195, miR-2861, miR-145, miR-214, miR-34a, miR-200a, let-7d-3p, miR-30d-5p, miR-638, miR-203a-3p, miR-1914-5p, miR-521, miR-125b, miR-370, miR-218 and miR-100). CONCLUSION: Therefore, defining promising circulating miRNAs or specific miRNA signatures of biological fluid samples can be useful for the screening, diagnosis, prognosis and clinical monitoring of women undergoing cervical carcinogenesis, but greater standardization of studies seems to be necessary for greater consolidation of information.

Properties and Application of Cell-Free DNA as a Clinical Biomarker.

Biomarkers are valuable tools in clinical practice. In 2001, the National Institutes of Health (NIH) standardized the definition of a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker has clinical relevance when it presents precision, standardization and reproducibility, suitability to the patient, straightforward interpretation by clinicians, and high sensitivity and/or specificity by the parameter it proposes to identify. Thus, serum biomarkers should have advantages related to the simplicity of the procedures and to the fact that venous blood collection is commonplace in clinical practice. We described the potentiality of cfDNA as a general clinical biomarker and focused on endothelial dysfunction. Circulating cell-free DNA (cfDNA) refers to extracellular DNA present in body fluid that may be derived from both normal and diseased cells. An increasing number of studies demonstrate the potential use of cfDNA as a noninvasive biomarker to determine physiologic and pathologic conditions. However, although still scarce, increasing evidence has been reported regarding using cfDNA in cardiovascular diseases. Here, we have reviewed the history of cfDNA, its source, molecular features, and release mechanism. We also show recent studies that have investigated cfDNA as a possible marker of endothelial damage in clinical settings. In the cardiovascular system, the studies are quite new, and although interesting, stronger evidence is still needed. However, some drawbacks in cfDNA methodologies should be overcome before its recommendation as a biomarker in the clinical setting.

Cyclic stretch-induced Crp3 sensitizes vascular smooth muscle cells to apoptosis during vein arterialization remodeling.

Vein graft failure limits the long-term patency of the saphenous vein used as a conduit for coronary artery bypass graft. Early graft adaptation involves some degree of intima hyperplasia to sustain the hemodynamic stress, but the progress to occlusion in some veins remains unclear. We have demonstrated that stretch-induced up-regulation of cysteine and glycine-rich protein 3 (Crp3) in rat jugular vein and human saphenous vein in response to arterialization. Here, we developed a Crp3-KO rat to investigate the role of Crp3 in vascular remodeling. After 28 days jugular vein arterialization, the intima layer was 3-fold thicker in the Crp3-KO that showed comparable smooth muscle cells (SMC) proliferation but an absence of early apoptosis observed in the wild-type rat (WT). We then investigated the role of Crp3 in early integrin-mediated signaling apoptosis in isolated jugular SMC. Interestingly, under basal conditions, ceramide treatment failed to induce apoptosis in both WT and Crp3-KO SMC. Under stretch, Crp3 expression increased in WT SMC and ceramide induced apoptosis. Immunoblotting analysis indicated that ceramide stretch-induced apoptosis in SMC is accompanied by a decrease in the phosphorylation status of both Fak and Akt, leading to an increase in Bax expression and caspase-3 cleavage. In contrast, ceramide failed to decrease Fak and Akt phosphorylation in Crp3-KO SMC and, therefore, there was no downstream induction of Bax expression and effector caspase-3 cleavage. Taken together, we provide evidence that stretch-induced Crp3 modulates vein remodeling in response to arterialization by sensitizing SMC to apoptosis.

Functional role of glucose metabolism, osmotic stress, and sodium-glucose cotransporter isoform-mediated transport on Na+/H+ exchanger isoform 3 activity in the renal proximal tubule.

Na(+)-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na(+)-H(+) exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.

Variation of mechanical properties and quantitative proteomics of VSMC along the arterial tree.

Vascular smooth muscle cells (VSMCs) are thought to assume a quiescent and homogeneous mechanical behavior after arterial tree development phase. However, VSMCs are known to be molecularly heterogeneous in other aspects and their mechanics may play a role in pathological situations. Our aim was to evaluate VSMCs from different arterial beds in terms of mechanics and proteomics, as well as investigate factors that may influence this phenotype. VSMCs obtained from seven arteries were studied using optical magnetic twisting cytometry (both in static state and after stretching) and shotgun proteomics. VSMC mechanical data were correlated with anatomical parameters and ultrastructural images of their vessels of origin. Femoral, renal, abdominal aorta, carotid, mammary, and thoracic aorta exhibited descending order of stiffness (G, P < 0.001). VSMC mechanical data correlated with the vessel percentage of elastin and amount of surrounding extracellular matrix (ECM), which decreased with the distance from the heart. After 48 h of stretching simulating regional blood flow of elastic arteries, VSMCs exhibited a reduction in basal rigidity. VSMCs from the thoracic aorta expressed a significantly higher amount of proteins related to cytoskeleton structure and organization vs. VSMCs from the femoral artery. VSMCs are heterogeneous in terms of mechanical properties and expression/organization of cytoskeleton proteins along the arterial tree. The mechanical phenotype correlates with the composition of ECM and can be modulated by cyclic stretching imposed on VSMCs by blood flow circumferential stress.

Short-term mechanical stretch fails to differentiate human adipose-derived stem cells into cardiovascular cell phenotypes.

BACKGROUND: We and others have previously demonstrated that adipose-derived stem cells (ASCs) transplantation improve cardiac dysfunction post-myocardium infarction (MI) under hemodynamic stress in rats. The beneficial effects appear to be associated with pleiotropic factors due to a complex interplay between the transplanted ASCs and the microenvironment in the absence of cell transdifferentiation. In the present work, we tested the hypothesis that mechanical stretch per se could change human ASCs (hASCs) into cardiovascular cell phenotypes that might influence post-MI outcomes. METHODS: Human ASCs were obtained from patients undergoing liposuction procedures. These cells were stretched 12%, 1Hz up to 96 hours by using Flexercell 4000 system. Protein and gene expression were evaluated to identify cardiovascular cell markers. Culture medium was analyzed to determine cell releasing factors, and contraction potential was also evaluated. RESULTS: Mechanical stretch, which is associated with extracellular signal-regulated kinase (ERK) phosphorylation, failed to induce the expression of cardiovascular cell markers in human ASCs, and mesenchymal cell surface markers (CD29; CD90) remained unchanged. hASCs and smooth muscle cells (SMCs) displayed comparable contraction ability. In addition, these cells demonstrated a profound ability to secrete an array of cytokines. These two properties of human ASCs were not influenced by mechanical stretch. CONCLUSIONS: Altogether, our findings demonstrate that hASCs secrete an array of cytokines and display contraction ability even in the absence of induction of cardiovascular cell markers or the loss of mesenchymal surface markers when exposed to mechanical stretch. These properties may contribute to beneficial post-MI cardiovascular outcomes and deserve to be further explored under the controlled influence of other microenvironment components associated with myocardial infarction, such as tissue hypoxia.

MicroRNA as a promising molecular biomarker in the diagnosis of breast cancer.

Introduction: Breast cancer represents the most prevalent malignancy among women. Recent advancements in translational research have focused on the identification of novel biomarkers capable of providing valuable insights into patient outcomes. Furthermore, comprehensive investigations aimed at discovering novel miRNAs, unraveling their biological functions, and deciphering their target genes have significantly contributed to our understanding of the roles miRNAs play in tumorigenesis. Consequently, these investigations have facilitated the way for the development of miRNA-based approaches for breast cancer prognosis, diagnosis, and treatment. However, conducting a more extensive array of studies, particularly among diverse ethnic groups, is imperative to expand the scope of research and validate the significance of miRNAs. This study aimed to assess the expression patterns of circulating miRNAs in plasma as a prospective biomarker for breast cancer patients within a population primarily consisting of individuals from Black, Indigenous, and People of Color (BIPOC) communities. Methods: We evaluated 49 patients with breast cancer compared to 44 healthy women. Results and discussion: All miRNAs analyzed in the plasma of patients with breast cancer were downregulated. ROC curve analysis of miR-21 (AUC = 0.798, 95% CI: 0.682-0.914, p <0.0001), miR-1 (AUC = 0.742, 95% CI: 0.576-0.909, p = 0.004), miR-16 (AUC = 0.721, 95% CI: 0.581-0.861, p = 0.002) and miR-195 (AUC = 0.672, 95% CI: 0.553-0.792, p = 0.004) showed better diagnostic accuracy in discrimination of breast cancer patients in comparison with healthy women. miR-210, miR-21 showed the highest specificities values (97.3%, 94.1%, respectively). Following, miR-10b and miR-195 showed the highest sensitivity values (89.3%, and 77.8%, respectively). The panel with a combination of four miRNAs (miR-195 + miR-210 + miR-21 + miR-16) had an AUC of 0.898 (0.765-0.970), a sensitivity of 71.4%, and a specificity of 100.0%. Collectively, our results highlight the miRNA combination in panels drastically improves the results and showed high accuracy for the diagnosis of breast cancer displaying good sensitivity and specificity.

Cell-Free Nuclear and Mitochondrial DNA as Potential Biomarkers for Assessing Sepsis Severity.

Sepsis continues to be a significant public health challenge despite advances in understanding its pathophysiology and management strategies. Therefore, this study evaluated the value of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) for assessing the severity and prognosis of sepsis. Ninety-four patients were divided into three groups: infection (n = 32), sepsis (n = 30), and septic shock (n = 32). Plasma samples were collected at the time of diagnosis, and cfDNA concentrations were determined by qPCR assay. The results showed that plasma cfDNA levels increased with the severity of the disease. To distinguish between patients with infection and those with sepsis, the biomarker L1PA290 achieved the highest AUC of 0.817 (95% CI: 0.725-0.909), demonstrating a sensitivity of 77.0% and a specificity of 79.3%. When cf-nDNA was combined with the SOFA score, there was a significant improvement in the AUC (0.916 (0.853-0.979)), sensitivity (88.1%), and specificity (80.0%). Moreover, patients admitted to the ICU after being diagnosed with sepsis had significantly higher cf-nDNA concentrations. In patients admitted to the ICU, combining cf-nDNA with the SOFA score yielded an AUC of 0.753 (0.622-0.857), with a sensitivity of 95.2% and a specificity of 50.0%. cfDNA can differentiate between patients with infection and those with sepsis. It can also identify patients who are likely to be admitted to the ICU by predicting those with indications for intensive care, suggesting its potential as a biomarker for sepsis.

Prognostication of OCT4 isoform expression in prostate cancer.

Cancer stem cells (CSCs) refer to a subset of tumor cells that self-renew and affect tumor heterogeneity. This model has attracted considerable interest in recent years due to its implications in the prognosis and clinical management of cancer because CSCs mediate the occurrence, growth, and recurrence of tumors. OCT4 is central to embryonic stem cell self-renewal and differentiation into specific lineages and encodes two chief isoforms that are generated by alternative splicing--OCT4A and OCT4B. Their function in prostate cancer (PCa) is unknown. The prognostic function of OCT4 isoforms in PCa samples was examined by immunohistochemistry (IHC) and sensitivity and specificity of the antibodies used were evaluated by molecular biology techniques. Biochemical and pathological data and specimens from 193 patients with PCa were evaluated retrospectively. IHC, western blot, immunofluorescence, and automated image analysis were also performed. IHC was performed on a tissue microarray, and western blot and immunofluorescence were performed using the PCa cell line DU-145. IHC expression of OCT4 isoforms correlated with biochemical and pathological parameters, particularly biochemical recurrence-free survival (BCRFS). Patients with higher levels of OCT4B had lower Gleason scores and decreased likelihood of experiencing biochemical recurrence (BR). OCT4A(+) OCT4B(-) patients had the shortest BCRFS, and positivity for OCT4B expression was an independent prognostic factor for BCRFS in the multivariate analysis. We conclude that the expression of OCT4B is a strong marker of good prognosis, and its presence is associated with a decreased likelihood of BR. Thus, OCT4B might represent a powerful clinical prognostic biomarker for PCa patients.

MicroRNA as potential biomarker for severity, progression, and therapeutic monitoring in animal models of limb-girdle muscular dystrophy: a systematic review.

Limb-girdle muscular dystrophies (LGMD) constitute a heterogeneous group of neuromuscular disorders in which there are alterations in proteins responsible for the preservation of muscle architecture and function, leading to proximal and progressive muscle weakness. There is, however, significant phenotypic and genotypic variation, as well as difficulty in establishing biomarkers that help to define pathogenic mechanisms and assess disease severity and progression. In this field, there is special attention to microRNAs, small non-coding RNA molecules related to the regulation of gene expression and, consequently, the production of proteins. Thus, this research aimed to verify the correlation between the expression of microRNAs and the severity, progression, and therapeutic response of LGMD animal models. A search was carried out in the PubMed, Embase, Scopus, ScienceDirect, Cochrane, and SciELO databases, with articles in English and without a time limit. The PRISMA 2020 checklist was used, and the protocol of this review was submitted to PROSPERO. The bibliographic survey of the 434 records found that 5 original articles met the inclusion criteria. The studies explored myomicroRNAs or miRNA panels with gene expression analysis. The analysis demonstrates that miR-1, 133a, and 206 are differentially expressed in serum and muscle. They change according to the degree of inflammation, fibrosis, muscle regeneration, and progression of the dystrophic process. MicroRNAs are up-regulated in dystrophic muscles, which are reversed after treatment in a dose-dependent manner. The present study inferred that miRs are essential in severity, progression, and therapeutic response in LGMD models and may be a useful biomarker in clinical research and prognosis. However, the practical application of these findings should be further explored.

Near-Infrared Spectroscopy with Supervised Machine Learning as a Screening Tool for Neutropenia.

The use of non-invasive tools in conjunction with artificial intelligence (AI) to detect diseases has the potential to revolutionize healthcare. Near-infrared spectroscopy (NIR) is a technology that can be used to analyze biological samples in a non-invasive manner. This study evaluated the use of NIR spectroscopy in the fingertip to detect neutropenia in solid-tumor oncologic patients. A total of 75 patients were enrolled in the study. Fingertip NIR spectra and complete blood counts were collected from each patient. The NIR spectra were pre-processed using Savitzky-Golay smoothing and outlier detection. The pre-processed data were split into training/validation and test sets using the Kennard-Stone method. A toolbox of supervised machine learning classification algorithms was applied to the training/validation set using a stratified 5-fold cross-validation regimen. The algorithms included linear discriminant analysis (LDA), logistic regression (LR), random forest (RF), multilayer perceptron (MLP), and support vector machines (SVMs). The SVM model performed best in the validation step, with 85% sensitivity, 89% negative predictive value (NPV), and 64% accuracy. The SVM model showed 67% sensitivity, 82% NPV, and 57% accuracy on the test set. These results suggest that NIR spectroscopy in the fingertip, combined with machine learning methods, can be used to detect neutropenia in solid-tumor oncology patients in a non-invasive and timely manner. This approach could help reduce exposure to invasive tests and prevent neutropenic patients from inadvertently undergoing chemotherapy.

The Role of miRNAs in the Prognosis of Triple-Negative Breast Cancer: A Systematic Review and Meta-Analysis.

Breast cancer is one of the most common malignancies among women around the world. The basal or triple-negative subtype (TNBC) is a heterogeneous group of tumors, characterized by its aggressive and metastatic nature, with low survival and worse prognosis. Research on genetic biomarkers, such as microRNAs (miRs) in TNBC, demonstrate their relevance in the prognosis of the disease. Therefore, the objective of this research was to verify the role of miRs in the prognosis of TNBC. A search was carried out in the PubMed (MEDLINE), Web of Science, and Scopus databases, with articles in the English language from 2010 to 2022. Only articles that analyzed the role of miRNAs in the prognosis of TNBC and that met the criteria of the MOOSE method were included. For the preparation and planning of this systematic review, a PRISMA checklist and the MOOSE method were used. The Newcastle-Ottawa Scale was used to analyze the quality of the included studies. The excluded criteria considered were: (1) studies that presented duplication in the databases; (2) reviews of the literature, clinical case reports, meta-analyses, conference abstracts, letters to the editor, theses, dissertations, and book chapters; (3) studies that stratified only women diagnosed with other subtypes of breast cancer subtypes; (4) experiments without a control or comparison group. After the bibliographic survey of the 2.274 articles found, 43 articles met the inclusion criteria, totaling 5421 patients with TNBC analyzed for this review. Six miRs (miR-155, miR-21, miR-27a/b/, miR-374a/b, miR-30a/c/e, and miR-301a) were included in the meta-analysis. A low expression of miR-155 was associated with reduced overall survival (OS) (HR: 0.68, 95% CI: 0.58-0.81). A high expression of miR-21 was a predictor of OS reduction (HR: 2.56; 95% CI: 1.49-4.40). In addition, high levels of miR-27a/b and miR-301a/b were associated with lower OS, while the decreased expression levels of miR-30 and miR-374a/b were associated with worse relapse-free survival (RFS) and shorter disease-free survival (DFS), respectively. The present study revealed that miRs play essential roles in the development of metastases, in addition to acting as suppressors of the disease, thus improving the prognosis of TNBC. However, the clinical application of these findings has not yet been investigated.

Rapid diagnosis of COVID-19 using FT-IR ATR spectroscopy and machine learning.

Early diagnosis of COVID-19 in suspected patients is essential for contagion control and damage reduction strategies. We investigated the applicability of attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy associated with machine learning in oropharyngeal swab suspension fluid to predict COVID-19 positive samples. The study included samples of 243 patients from two Brazilian States. Samples were transported by using different viral transport mediums (liquid 1 or 2). Clinical COVID-19 diagnosis was performed by the RT-PCR. We built a classification model based on partial least squares (PLS) associated with cosine k-nearest neighbours (KNN). Our analysis led to 84% and 87% sensitivity, 66% and 64% specificity, and 76.9% and 78.4% accuracy for samples of liquids 1 and 2, respectively. Based on this proof-of-concept study, we believe this method could offer a simple, label-free, cost-effective solution for high-throughput screening of suspect patients for COVID-19 in health care centres and emergency departments.

Physiological and Biochemical Vascular Reactivity Parameters of Angiotensin II and the Action of Biased Agonist TRV023.

Vascular reactivity experiments using isolated aortic rings have been widely used as a model for physiological and pharmacological studies since the early sixties. Here, we suggest several parameters that the researcher should pay attention to when investigating angiotensin II in their experimental models. Angiotensin II is one of the active peptides of the renin-angiotensin system and exerts its effect through the AT1 and AT2 receptors. Some studies seek to understand the effects of angiotensin II receptors at the vascular level by using vascular reactivity experiments. However, because of the large number of variations, there are only a handful of reactivity studies that seek to use this method. Thus, the objective of this study was to standardize experimental methods with angiotensin II, through vascular reactivity protocols. For this, variables such as basal tension, concentration interval, single concentration, curve concentration response, and multiple experiments using the same aortic ring were developed using the technique of vascular reactivity in an organ bath. This is the first study that has standardized the vascular reactivity protocol. In addition, we demonstrated the effects of TRV023-biased ligand of the AT1R at vascular sites.

Activation of Interleukin-1 Beta in Arterialized Vein Grafts and the Influence of the -511C/T IL-1ß Gene Polymorphism.

The interleukin-1 family is associated with innate immunity and inflammation. The latter has been linked to the genesis of cardiovascular diseases. We, therefore, investigated whether interleukin-1 beta (IL-1ß) is activated during arterialization of vein grafts. First, we examined the activation of IL-1ß using the rat arterialized jugular vein serially sampled for up to 90 days. IL-1ß expression increased 18 times on day 1 in the arterialized rat jugular vein and remained five times above nonarterialized vein levels for up to 90 days. Similarly, IL-1ß expression increased early (1-5 days) in human vein graft autopsy samples compared with late phases (1-4 years). Activation was also detected in ex vivo arterialized human saphenous veins. Upon stratification of the results, we uncovered a T allele promoter attenuating effect in IL-1ß activation in response to hemodynamic stress. Altogether, the results show that IL-1ß is activated during arterialization of vein grafts in rats and humans, and this response is modulated by -511C/T IL-1ß gene polymorphism. It is tempting to speculate that the activation of IL-1ß, and consequently local inflammation, modulates early vascular remodeling and that the gene polymorphism may be useful in predicting outcomes or assisting in interventions.

Porcine adipose tissue-derived mesenchymal stem cells retain their proliferative characteristics, senescence, karyotype and plasticity after long-term cryopreservation.

We and others have provided evidence that adipose tissue-derived mesenchymal stem cells (ASCs) can mitigate rat cardiac functional deterioration after myocardial ischemia, even though the mechanism of action or the relevance of these findings to human conditions remains elusive. In this regard, the porcine model is a key translational step, because it displays heart anatomic-physiological features that are similar to those found in the human heart. Towards this end, we wanted to establish the cultural characteristics of porcine ASCs (pASCs) with or without long-term cryostorage, considering that allogeneic transplantation may also be a future option. Compared to fresh pASCs, thawed cells displayed 90-95% viability and no changes in morphological characteristics or in the expression of surface markers (being pASCs characterized by positive markers CD29(+); CD90(+); CD44(+); CD140b(+); CD105(+); and negative markers CD31(-); CD34(-); CD45(-) and SLA-DR(-); n = 3). Mean population doubling time was also comparable (64.26±15.11 hours to thawed cells vs. 62.74±18.07 hours to fresh cells) and cumulative population doubling increased constantly until Passage 10 (P10) in the entire cell population, with a small and gradual increase in senescence (P5, 3.25%±0.26 vs. 3.47%±0.32 and P10, 9.6%±0.29 vs. 10.67%±1.25, thawed vs. fresh; SA-ß-Gal staining). Chromosomal aberrations were not observed. In addition, under both conditions pASCs responded to adipogenic and osteogenic chemical cues in vitro. In conclusion, we have demonstrated the growth characteristics, senescence, and the capacity of pASCs to respond to chemical cues in vitro and have provided evidence that these properties are not influenced by cryostorage in 10% DMSO solution.

ACE as a mechanosensor to shear stress influences the control of its own regulation via phosphorylation of cytoplasmic Ser(1270).

OBJECTIVES: We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser(1270) are involved in shear-stress (SS)-induced downregulation of the enzyme. METHODS AND RESULTS: Western blotting analysis showed that SS (18 h, 15 dyn/cm(2)) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser(1270) compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor. CONCLUSIONS: ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser(1270), consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser(1270).

Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather than shear stress.

AIMS: Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein-protein interaction with actin filaments in the heart and is involved in muscle differentiation and vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization in human and rat veins. METHODS AND RESULTS: Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT-PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1-14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28-90 days) it appeared more scattered in the vessel layers. CONCLUSION: Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.

Identificação e caracterização de proteínas modificadas em enxertos de veias safenas humanas arterializadas no modelo ex vivo / Identification and characterization of modified proteins in arterialized human saphenous vein using an ex vivo system

A revascularização cardíaca utilizando a ponte de safena é um procedimento bastante utilizado para restabelecer o fluxo coronariano. Apesar do sucesso deste procedimento, a patência deste enxerto pode chegar a menos de 50% em 10 anos. Atribui-se parte deste insucesso a variações no processo adaptativo à nova condição hemodinâmica, onde o shear stress e o estiramento aumentados podem estar interferindo na função endotelial e vascular. Este processo envolve a participação de diversas proteínas e o estudo de como elas participam conjuntamente é uma importante abordagem para entender as alterações fisiológicas e patológicas que ocorrem no enxerto vascular. Neste trabalho, tecnologias proteômicas, gel 2-D e ICAT, foram utilizadas para identificar as proteínas que são modificadas nas fases precoces da arterialização do enxerto venoso. Foi utilizado um sistema ex vivo de perfusão controlada, desenvolvido em nosso laboratório, onde a veia safena humana foi cultivada tanto em regime hemodinâmico venoso (5 mL/min) e arterial (50 mL/min, 80 mmHg) por 24 horas. Dentre as proteínas identificadas, a maioria apresenta funções estruturais como, por exemplo, -actina de músculo liso, CRP1, colágeno VI, tropomiosina, miosina, desmina e vimentina. Para avaliação funcional foram selecionadas a -SMA e a CRP. A -SMA mostrou-se diminuída nas fases mais precoces da arterialização venosa, com quase desaparecimento após 3 dias da cirurgia, seguido de um aumento nos períodos subseqüentes. A CRP3 mostrou-se com expressão predominantemente arterial tanto em amostra humana como de rato. A arterialização de segmentos venosos induziu a expressão da CRP3, sendo dependente do aumento do estiramento (stretch) nas células musculares lisas e não do aumento do shear stress na superfície endotelial. Coletivamente, neste trabalho caracterizamos duas proteínas que foram modificadas durante o processo de arterialização e/ou adaptação da veia à condição hemodinâmica arterial...

Coronary artery bypass surgery by saphenous vein graft is still widely used to revascularization of ischemic heart. Despite the success of this procedure, about 50% occlude after 5-10 years. The vein graft is subjected to increased tensile stress and the adaptive vein response to the arterial hemodynamic condition may predispose to bypass occlusion. Several proteins are modulated during arterialization, the understanding of the molecular changes of this process may be useful to new therapeutics approaches development attempting to increase vein graft patency. In this work, proteomics plataform, gel 2-D and ICAT, were used to identify the proteins that are modified in the early stages of vein graft rterialization. Human saphenous vein were cultured in an ex vivo flow through system in both venous (5 ml / min) and arterial (50 ml / min, 80 mm Hg) hemodymanic conditions for 24 hours. The identified proteins were related to cell structural function, such as -SMA, CRP1, collagen VI, tropomyosin, myosin, desmin and vimentin. To functional characterization, -SMA and CRP were selected. In rat vein arterialization model, - SMA showed to be decreased during the early stages of arterialization and almost disappeared after 3 days of surgery. Later on, -SMA-positive cells increase reaching similar expression levels of normal jugular vein. The expressiom of CRP3 showed to be predominantly to arterial beds both in human and rat. When vein segment were submitted to arterial hemodynamic condition, it was observed a significant induction of CRP3 expression. Interestingly, the increase of CRP3 is dependent of stretch stimulus in smooth muscle cells while shear stress did not modify its expression in endothelial cells. Collectively, we successfully identified proteins differentially expressed during the vein arterialization by using proteomic technique. -SMA and CRP3 were modified in vein segments exposed to arterial hemodynamic condition and efficiently discriminate...