Cristina Cuesta

Identification of bone morphogenetic protein 9 (BMP9) as a novel profibrotic factor in vitro.

Upregulated synthesis of extracellular matrix (ECM) proteins by myofibroblasts is a common phenomenon in the development of fibrosis. Although the role of TGF-β in fibrosis development has been extensively studied, the involvement of other members of this superfamily of cytokines, the bone morphogenetic proteins (BMPs) in organ fibrosis has given contradictory results. BMP9 is the main ligand for activin receptor-like kinase-1 (ALK1) TGF-β1 type I receptor and its effect on fibrosis development is unknown. Our purpose was to study the effect of BMP9 in ECM protein synthesis in fibroblasts, as well as the involved receptors and signaling pathways. In cultured mice fibroblasts, BMP9 induces an increase in collagen, fibronectin and connective tissue growth factor expression, associated with Smad1/5/8, Smad2/3 and Erk1/2 activation. ALK5 inhibition with SB431542 or ALK1/2/3/6 with dorsomorphin-1, inhibition of Smad3 activation with SIS3, and inhibition of the MAPK/Erk1/2 with U0126, demonstrates the involvement of these pathways in BMP9-induced ECM synthesis in MEFs. Whereas BMP9 induced Smad1/5/8 phosphorylation through ALK1, it also induces Smad2/3 phosphorylation through ALK5 but only in the presence of ALK1. Summarizing, this is the first study that accurately identifies BMP9 as a profibrotic factor in fibroblasts that promotes ECM protein expression through ALK1 and ALK5 receptors.

Role of 5-HT7 receptors in the inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats.

The role of calcitonin gene-related peptide (CGRP) in the modulation of vascular tone has been widely documented. Indeed, electrical stimulation of the perivascular sensory outflow in pithed rats induces vasodepressor responses by activation of CGRP receptors. This study investigated the role of 5-HT7 receptors in the inhibition of the rat vasodepressor sensory outflow. Male Wistar pithed rats were pretreated with i.v. continuous infusions of hexamethonium and methoxamine, followed by physiological saline or AS-19 (a 5-HT7 receptor agonist). Then, electrical stimulation of the spinal cord resulted in frequency-dependent decreases in DBP. The infusions of AS-19, as compared to those of saline, inhibited the vasodepressor responses induced by electrical stimulation without affecting those to i.v. bolus injections of exogenous α-CGRP. This inhibition by AS-19 was abolished by the antagonists pimozide (5-HT7) or sulfisoxazole (ETA), but not by indomethacin (COX1/2) or losartan (AT1), at doses that did not affect per se the electrically-induced vasodepressor responses. Interestingly, glibenclamide (an ATP-dependent K(+) channel blocker) attenuated these vasodepressor responses. The present results suggest that AS-19-induced inhibition of the rat vasodepressor sensory CGRPergic outflow is mainly mediated by 5-HT7 receptors via endothelin release, with the possible involvement of ATP-dependent K(+) channels.

Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

The involvement of Ras‐GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H‐ and N‐Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K‐Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K‐Ras knockout (KO) mouse embryonic fibroblasts (K‐ras−/−) stimulated with transforming growth factor‐β1 (TGF‐β1) exhibited reduced proliferation and impaired mobility than wild‐type fibroblasts. Moreover, an increase on ECM production was observed in K‐Ras KO fibroblasts in basal conditions. The absence of K‐Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF‐β1‐induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K‐ras but reduced migration and ECM proteins expression only in wild‐type fibroblasts, while the PI3K‐AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K‐Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K‐Ras may be a crucial mediator in TGF‐β1‐mediated effects in this cell type. J. Cell. Physiol. 231: 2224–2235, 2016.

Overexpression of the short endoglin isoform reduces renal fibrosis and inflammation after unilateral ureteral obstruction.

Transforming growth factor beta 1 (TGF-β1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-β1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG(+)) and we have performed an UUO in S-ENG(+) and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG(+) mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG(+) mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.