bioRxiv Science⌕ Search

Biology subjects

Fresno, M.

Publications and source records attributed to Fresno, M..

4 recordsLinked to original sources

Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

Rho GTPases are molecular targets of bacterial toxins that modulate their enzymatic activity. RhoA, RhoB and RhoC are almost identical and play critical roles in generating actomyosin-mediated contractile forces that cause endothelial hyperpermeability during inflammation. Searching for new treatments to modulate endothelial integrity, we demonstrate that the specific and simultaneous activation of these three Rho GTPases with a chimeric recombinant toxin does not induce cell contraction but enhances homeostatic endothelial barrier function, increases reticular adherens junctions and preserves the microvascular endothelium in response to pathological inflammatory challenges in vitro and in vivo. This pro-barrier effect is specifically mediated by RhoC, whose activity is increased by cell confluence. The uniqueness of RhoC relies on an arginine 188 within its hypervariable region that determines its junctional localization, high homeostatic activity, and barrier-protective function. Quantitative proteomics revealed that RhoC regulates the expression of myosin light chain proteins and junction-stabilizing actomyosin. Thus, harnessing the activity of RhoC represents a potential therapy for strengthening endothelial barriers during pathological inflammation.

cell biology↗

Anti-inflammatory immunomodulation for the treatment of Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a rare disease where the diaphragm does not develop properly altering lung development with no established therapy. We have analyzed the effect of anti-inflammatory immunomodulators that influence macrophage activation in animal CDH models. In the widely-used nitrofen-induced model of CDH in pregnant rats, administration of a single dose of atypical Toll-like Receptors TLR2/4 dual ligands (CS1 and CS2), 3 days after nitrofen, cured diaphragmatic hernia in 73 % of the fetuses, repaired the lesion with complete diaphragm closure. Moreover, they also improve pulmonary hypoplasia and vessel hypertrophy, enhancing pulmonary maturity of fetuses. CS1 treatment also rescued the CDH phenotype in the G2-GATA4Cre;Wt1fl/fl CDH genetic mice model. Only 1 out 11 mutant embryos showed CDH after CS1 administration, whereas CDH prevalence was 70% in untreated mutant embryos. Mechanistically, CS1 stimulated the infiltration of repairing M2 macrophages (CD206+ and Arg1+) into the damaged diaphragm and reduced T cell infiltration. Alteration in retinoic acid pathways a have been also implicated in the etiology of CDH. TLR2/4 dual ligands also induced retinol pathway genes, including RBP1, RALDH2, RAR and RAR{beta}, in the affected lungs and the diaphragm and in macrophages in vitro. The present results place atypical TLR2/4 ligands as a promising solution for CDH, where the own immune system of the fetus is responsible for repairing the hernia/damage in the diaphragm, ensuring the correct positioning and development of all organs.

immunology↗

Balance of AKT isoforms in intestinal barrier function. Implications for IBD therapy.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/547629v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1716da6org.highwire.dtl.DTLVardef@1d5b63corg.highwire.dtl.DTLVardef@1af34a6org.highwire.dtl.DTLVardef@1a993ab_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOWORKING MODELC_FLOATNO C_FIG The leakiness of the intestinal epithelial barrier plays a major role in the development of inflammatory bowel disease (IBD). We show that either Akt1 overexpression or Akt2 chemical inhibition have similar effects on intestinal epithelial cells, enhancing the permeability of the epithelial barrier and affecting the expression and/or intracellular localization of the tight junctions proteins (TJP) whereas Akt1 inhibition or Akt2 overexpression show the opposite results. Akt1 overexpression or Akt2 inhibition also promotes activation of {beta}-Catenin and Lgr5. Moreover, mouse intestinal organoids treated ex vivo with Akt1 inhibitors present dissembled and disorganized structures that mimic IBD histological features. Importantly, in DSS-induced IBD in mice, Akt2 inhibition strongly ameliorates disease with animals presenting healthy-like crypts, having tightened colon epithelial cells expressing TJP, and presenting an anti-inflammatory M2 macrophage phenotype. In contrast, treatment with Akt1 inhibitors enhances histological damage, TJP disorganization reduces Lgr5+ expression and promotes a more proinflammatory milieu further favoring the development of the disease. Those results suggest that a balance between Akt1 and Akt2 regulates the functionality of the intestinal epithelial barrier, cell renewal and inflammation and that Akt2 inhibition can be considered as a new therapy for IBD. THE PAPER EXPLAINEDInflammatory bowel disease (IBD), that includes Croh[n]s disease and ulcerative colitis, needs better therapeutic options. Importantly, IBD is a risk factor for the development of colorectal cancer. IBD is associated with epithelial barrier dysfunction, decreasing its permeability, and finally leading to chronic inflammation and disease. We show that the Akt1 and Akt2 protein kinase isoforms play different and opposite roles in the integrity of intestinal epithelial barrier by affecting the expression and/or the intracellular localization of tight junction proteins (TJP) responsible for barrier formation and reducing anti-barrier ones when Akt1 is overexpressed or Akt2 is inhibited. Those results were confirmed in ex vivo organoids, a system that mimics intestinal tissue organization, where Akt1 inhibition resulted in dissembled and disorganized structures that resembled those found in IBD patients. There is a balance between Akt1 and Akt2 activation on the epithelial cells, and when Akt1 is less abundant than Akt2, the barrier is disrupted by the dysregulation of the tightness of the barrier by the TJPs, leading also to an acute phase of inflammation. Epithelial cells are also incapable of renewing themselves by the absence of active Akt1, making the damage more severe, creating a pro-inflammatory niche that derives on chronic inflammation and severe IBD. Interestingly, those in vitro and ex vivo results can be translated to the whole animals. IBD disease and colon inflammation and histological alterations induced in mice by an irritant DSS could be either worsened with an Akt1 inhibitor or much improved with treatment with an Akt2 inhibitor, respectively. The inhibition of Akt2 may be considered as a treatment or co-treatment of IBD by regulating this balance to an Akt1 predominance environment. Thus, the use of Akt2 inhibitors, already developed, could be an alternative way to treat this disease.

immunology↗

Prostaglandin F2α-induced TGFβ-PMEPA1 pathway is a critical mediator of epithelial plasticity and ovarian carcinoma progression.

Prostaglandin (PG) F2 has been scarcely studied in cancer. We have identified a new role for PGF2 in ovarian cancer, stimulating the production of TGF{beta} and the consequent induction of PMEPA1. We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGF{beta} signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated to higher E-cadherin expression levels while {beta}-catenin nuclear translocation was inhibited. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.\n\nSignificanceThis work identifies Prostaglandin F2 as an inducer, through NFAT, of TGF{beta} and together with this up-regulate PMEPA1, which is identified both as a drug target and as a prognostic biomarker in ovarian tumors, potentially useful in clinical practice.

cancer biology↗