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Regular physical activity exerts cardiovascular protective effects in healthy individuals and those with chronic cardiovascular diseases. Exercise is accompanied by an increased plasma concentration of α-calcitonin gene-related peptide (αCGRP), a 37-amino acid peptide with vasodilatory effects and causative roles in migraine. Moreover, mouse models revealed that loss of αCGRP disrupts physiological adaptation of the cardiovascular system to exercise in normotension and aggravates cardiovascular impairment in primary chronic hypertension, both can be reversed by αCGRP administration. This suggests that αCGRP agonists could be a therapeutic option to mediate the cardiovascular protective effects of exercise in clinical setting where exercise is not possible or contraindicated. Of note, FDA has recently approved αCGRP antagonists for migraine prophylaxis therapy, however, the cardiovascular safety of long-term anti-CGRP therapy in individuals with cardiovascular diseases has yet to be established. Current evidence from preclinical models suggests that chronic αCGRP antagonism may abolish the cardiovascular protective effects of exercise in both normotension and chronic hypertension.

Figure S4. Actin cytoskeletal changes in vascular endothelial monolayers treated with RSPO3 for 6 h. Green, phalloidin staining of actin fibers; blue, DAPI staining of nuclei. Microphotographs were taken using a Zeiss Axioskope equipped with Axio-CamMRm and AxioVision Rel.4.6 software and are representative of three independent experiments run in triplicates. Original magnification, 630×. (PDF 4093 kb)

Figure S3. Assessment of barrier function in dermal microvascular endothelial monolayers. Uniform tight monolayers of HDMVEC cultured in stabilized and collagen coated 8W10E+ array chambers were treated with RSPO3, IL-1β or a combination of RSPO3 and IL-1β. (A) Original ECIS plot of resistance measurements (line graph) from one representative of three independent experiments run in duplicates at 4000 Hz (indicative of cell-cell adhesion tightness). (B) Bar graph showing the data of barrier function measurements continuously recorded for and at 12 h from three independent experiments run in duplicates. Black, vehicle; Green, IL-1β; Yellow, RSPO3; Purple, RSPO3 + IL-1β. Error bars are mean ± S.D. ***P

Figure S6. Induction of RSPO3 expression by IL-1β in human vascular endothelium. (A) Immunoblot of RSPO3 (31 kDa) in HCAEC treated with IL-1β for 24 h. In-gel stained 75 kDa band served as loading control and for immunoblot normalization in densitometric analysis. (B) RSPO3 expression levels quantified by densitometry analysis. Data are mean ± S.E.M of three independent experiments, **P

Figure S1. Confluent tight endothelial monolayer formation in 8W10E+ ECIS arrays. Immediately after cell seeding, resistance measurements (in Ohms) were started and are displayed as normalized resistance (subsequent values were divided by initial values). Increase in resistance with respect to time denotes that cells were forming contacts between each other. The steady state shows the stage at which maximum resistance is reached to form a tight monolayer. Resistance measurements were carried out in duplicate wells which were grouped and averaged to plot as single curve. Error bars represent mean ± S.E.M. Figures show the original plot of resistance measured at 4000 Hz (indicative of cell-cell adhesion tightness). Red, blue: record of monolayer formation of untreated (A) HCAEC and (B) HCMVEC grown in duplicate wells from two independent experiments. All treatments for subsequent assessment of barrier function were started after formation of stable tight monolayers. (PDF 636 kb)

Figure S5. β-catenin alignment at adherens junctions in vascular endothelial monolayers treated with RSPO3. Immunofluorescence staining of β-catenin (red) and DAPI staining of nuclei (blue) in HCAEC either untreated, or treated with RSPO3 for 6 h. Microphotographs were taken using a Zeiss Axioskope equipped with Axio-CamMRm and AxioVision Rel.4.6 software and are representative of three independent experiments run in triplicates. Original magnification, 630×. (PDF 5145 kb)

Supplementary methods. (PDF 124 kb)

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection. (VLID)4420919

Barrier function of Wnt5A-treated VEC in the presence or absence of WIF1 and sFRP1. a ECIS assisted measurements (Additional file 4: supplementary methods) showing resistance of HCAEC monolayers grown in 8W10E+ arrays treated with vehicle (black), Wnt5A (green), Wnt5A + WIF1 (yellow) and Wnt5A + sFRP1 (purple). Data shown are the resistance measurements conducted at 4000 Hz and are mean ± SEM of 2 wells from 1 out of three representative experiments. b Barrier function measurements indicating the significance of WIF1’s antagonistic effect on Wnt5A and are mean ± SEM of three independent experiments run with duplicate wells. *P

Supplementary methods. (PDF 300 kb)

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.

Expression of CD31 in HCAEC. Immunofluorescence staining for CD31 protein (red) in HCAEC either untreated (none) or treated with Wnt5A or TNF-alpha (20 U/mL) for 8 h. Nuclei are stained blue (DAPI). Zeiss Axioskope, original magnification 630 × . (PDF 8535 kb)

Supplementary methods. (PDF 300 kb)

Objective— Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. Approach and Results—We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Conclusions—Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.

Expression of CD31 in HCAEC. Immunofluorescence staining for CD31 protein (red) in HCAEC either untreated (none) or treated with Wnt5A or TNF-alpha (20 U/mL) for 8 h. Nuclei are stained blue (DAPI). Zeiss Axioskope, original magnification 630 × . (PDF 8535 kb)

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.