Rafael Dariolli

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.

Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to widespread motor neuron death, general palsy and respiratory failure. The most prevalent sporadic ALS form is not genetically inherited. Attempts to translate therapeutic strategies have failed because the described mechanisms of disease are based on animal models carrying specific gene mutations and thus do not address sporadic ALS. In order to achieve a better approach to study the human disease, human induced pluripotent stem cell (hiPSC)-differentiated motor neurons were obtained from motor nerve fibroblasts of sporadic ALS and non-ALS subjects using the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus system and submitted to microarray analyses using a whole human genome platform. DAVID analyses of differentially expressed genes identified molecular function and biological process-related genes through Gene Ontology. REVIGO highlighted the related functions mRNA and DNA binding, GTP binding, transcription (co)-repressor activity, lipoprotein receptor binding, synapse organization, intracellular transport, mitotic cell cycle and cell death. KEGG showed pathways associated with Parkinsons disease and oxidative phosphorylation, highlighting iron homeostasis, neurotrophic functions, endosomal trafficking and ERK signaling. The analysis of most dysregulated genes and those representative of the majority of categorized genes indicates a strong association between mitochondrial function and cellular processes possibly related to motor neuron degeneration. In conclusion, iPSC-derived motor neurons from motor nerve fibroblasts of sporadic ALS patients may recapitulate key mechanisms of neurodegeneration and may offer an opportunity for translational investigation of sporadic ALS. Large gene profiling of differentiated motor neurons from sporadic ALS patients highlights mitochondrial participation in the establishment of autonomous mechanisms associated with sporadic ALS.

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-β-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.

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

We previously demonstrated that uroguanylin (UGN) significantly inhibits Na(+)/H(+) exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK(1)). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H(+) secretion in rat renal proximal segments, through a H(+)-sensitive microelectrode. UGN (1 μM) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK(1) cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK(1) cells. Finally, treatment of LLC-PK(1) cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis.

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.

Dipeptidyl Peptidase IV Inhibition Exerts Renoprotective Effects in Rats with Established Heart Failure

Circulating dipeptidyl peptidase IV (DPPIV) activity is associated with worse cardiovascular outcomes in humans and experimental heart failure (HF) models, suggesting that DPPIV may play a role in the pathophysiology of this syndrome. Renal dysfunction is one of the key features of HF, but it remains to be determined whether DPPIV inhibitors are capable of improving cardiorenal function after the onset of HF. Therefore, the present study aimed to test the hypothesis that DPPIV inhibition by vildagliptin improves renal water and salt handling and exerts anti-proteinuric effects in rats with established HF. To this end, male Wistar rats were subjected to left ventricle (LV) radiofrequency ablation or sham operation. Six weeks after surgery, radiofrequency-ablated rats who developed HF were randomly divided into two groups and treated for 4 weeks with vildagliptin (120 mg/kg/day) or vehicle by oral gavage. Echocardiography was performed before (pretreatment) and at the end of treatment (post-treatment) to evaluate cardiac function. The fractional area change (FAC) increased (34 ± 5 vs. 45 ± 3%, p < 0.05), and the isovolumic relaxation time decreased (33 ± 2 vs. 27 ± 1 ms; p < 0.05) in HF rats treated with vildagliptin (post-treatment vs. pretreatment). On the other hand, cardiac dysfunction deteriorated further in vehicle-treated HF rats. Renal function was impaired in vehicle-treated HF rats as evidenced by fluid retention, low glomerular filtration rate (GFR) and high levels of urinary protein excretion. Vildagliptin treatment restored urinary flow, GFR, urinary sodium and urinary protein excretion to sham levels. Restoration of renal function in HF rats by DPPIV inhibition was associated with increased active glucagon-like peptide-1 (GLP-1) serum concentration, reduced DPPIV activity and increased activity of protein kinase A in the renal cortex. Furthermore, the anti-proteinuric effect of vildagliptin treatment in rats with established HF was associated with upregulation of the apical proximal tubule endocytic receptor megalin and of the podocyte main slit diaphragm proteins nephrin and podocin. Collectively, these findings demonstrate that DPPIV inhibition exerts renoprotective effects and ameliorates cardiorenal function in rats with established HF. Long-term studies with DPPIV inhibitors are needed to ascertain whether these effects ultimately translate into improved clinical outcomes.

Proteome analysis of acute kidney injury – Discovery of new predominantly renal candidates for biomarker of kidney disease ☆

The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are organ and process specific. Here, we have used different tissues from a controlled porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60min) and 4, 11 and 16h post-reperfusion, and renal cortex samples after 24h of reperfusion. Peptides were analyzed on the Q-Exactive™. In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighted by transcription factors and epithelial adherens junction proteins. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that may provide a set of targets for kidney injury. Additionally, we identified 49, being 4 predominantly renal, proteins in urine. As prove of concept, we validated one of the identified biomarkers, dipeptidyl peptidase IV, in a set of patients with diabetic nephropathy. In conclusion, we identified 55 systemic proteins, some of them predominantly renal, candidates for biomarkers of renal disease. BIOLOGICAL SIGNIFICANCE The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. Here we propose a comprehensive, sequential screening procedure able to identify and validate potential biomarkers for kidney disease, using kidney ischemia/reperfusion as a paradigm for a kidney pathological event.

Catheter‐based induction of renal ischemia/reperfusion in swine: description of an experimental model

Several techniques to induce renal ischemia have been proposed: clamp, PVA particles, and catheter‐balloon. We report the development of a controlled, single‐insult model of unilateral renal ischemia/reperfusion (I/R) without contralateral nephrectomy, using a suitable model, the pig. This is a balloon‐catheter‐based model using a percutaneous, interventional radiology procedure. One angioplasty balloon‐catheter was placed into the right renal artery and inflated for 120 min and reperfusion over 24 h. Serial serums were sampled from the inferior vena cava and urine was directly sampled from the bladder throughout the experiment, and both kidneys were excised after 24 h of reperfusion. Analyses of renal structure and function were performed by hematoxylin–eosin/periodic Acid‐Schiff, serum creatinine (SCr), blood urea nitrogen (BUN), fractional excretion of ions, and glucose, SDS‐PAGE analysis of urinary proteins, and serum neutrophil gelatinase‐associated lipocalin (NGAL). Total nitrated protein was quantified to characterize oxidative stress. Acute tubular necrosis (ATN) was identified in every animal, but only two animals showed levels of SCr above 150% of baseline values. As expected, I/R increased SCr and BUN. Fractional sodium, potassium, chloride, and bicarbonate excretion were modulated during ischemia. Serum‐nitrated proteins and NGAL had two profiles: decreased with ischemia and increased after reperfusion. This decline was associated with increased protein excretion during ischemia and early reperfusion. Altogether, these data show that the renal I/R model can be performed by percutaneous approach in the swine model. This is a suitable translational model to study new early renal ischemic biomarkers and pathophysiological mechanisms in renal ischemia.

Development of a closed-artery catheter-based myocardial infarction in pigs using sponge and lidocaine hydrochloride infusion to prevent irreversible ventricular fibrillation

The objectives of this study were to develop a robust, homogeneous, viable and inexpensive model of closed‐artery catheter‐based model of myocardial infarction (MI) in pigs without major cardiac dysfunction. Suitable animal models that mimic human cardiovascular conditions are of paramount importance to understand the effects of novel therapeutic strategies to improve tissue perfusion and prevent cardiac deterioration post‐MI. Pigs (N = 21, BW = 17 ± 1 kg) receiving continuous iv lidocaine hydrochloride were subjected to percutaneous intracoronary implant of foam sponge into the proximal left circumflex coronary artery. Intraprocedure mortality was 23.8%. ST segment elevation and increased serum Troponin T and CK‐MB were documented in all animals. Thirty days after occlusion, echocardiography (95% IC [9.3–12.4%]) and anatomopathological (95% CI [9.3–12.6%]) analyses confirmed a significant and reproducible MI. Taken together, we provide evidence for a suitable closed‐artery catheter‐based method to produce MI in pigs accompanied by tissue hypoperfusion and absence of overt heart failure.

Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway.

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.