Author response: Lymphangiogenic therapy prevents cardiac dysfunction by ameliorating inflammation and hypertension (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The lymphatic vasculature is involved in the pathogenesis of acute cardiac injuries, but little is known about its role in chronic cardiac dysfunction. Here, we demonstrate that angiotensin II infusion induced cardiac inflammation and fibrosis at 1 week and caused cardiac dysfunction and impaired lymphatic transport at 6 weeks in mice, while co-administration of VEGFCc156s improved these parameters. To identify novel mechanisms underlying this protection, RNA sequencing analysis in distinct cell populations revealed that VEGFCc156s specifically modulated angiotensin II-induced inflammatory responses in cardiac and peripheral lymphatic endothelial cells. Furthermore, telemetry studies showed that while angiotensin II increased blood pressure acutely in all animals, VEGFCc156s-treated animals displayed a delayed systemic reduction in blood pressure independent of alterations in angiotensin II-mediated aortic stiffness. Overall, these results demonstrate that VEGFCc156s had a multifaceted therapeutic effect to prevent angiotensin II-induced cardiac dysfunction by improving cardiac lymphatic function, alleviating fibrosis and inflammation, and ameliorating hypertension. Introduction The lymphatic vasculature plays an important role in mediating fluid balance and local inflammation in many tissues and organs throughout the body (Huang et al., 2017; Oliver et al., 2020). The heart also carries a complex lymphatic network and relies on cardiac lymphatics to drain interstitial fluids and immune cells for maintaining homeostasis (Brakenhielm and Alitalo, 2019). Disrupting cardiac lymphatics can cause myocardial edema and lead to impaired cardiac function (Cui et al., 2001; Mehlhorn et al., 1995), indicating a critical role of cardiac lymphatics in cardiovascular physiology. Recently, cardiac lymphatics have emerged as a new therapeutic target for acute cardiac injuries. Several studies have demonstrated using rodent models that therapeutic lymphangiogenesis reduced myocardial edema and fibrosis, resolved inflammation, and improved cardiac function after myocardial infarction (Henri et al., 2016; Houssari et al., 2020; Klotz et al., 2015; Tatin et al., 2017; Trincot et al., 2019; Vieira et al., 2018) or ischemia and reperfusion injury (Shimizu et al., 2018). These studies in aggregate concluded that the improvement in cardiac function was due to local proliferation of lymphatic vessels at the site of injury in response to lymphangiogenic therapies. Importantly, these animal models represent cardiac injuries, in which blood and lymphatic vessels are surgically manipulated. However, the role of the lymphatic vasculature in the pathogenesis of chronic cardiac dysfunction and the effects of lymphangiogenic therapies on cardiac dysfunction remain to be explored. Here, we explored the role of the lymphatic vasculature in cardiac dysfunction and investigated the effects of a lymphangiogenic therapy (VEGFCc156s) on the angiotensin II infusion-induced cardiac dysfunction model. We found that angiotensin II infusion-induced cardiac dysfunction is associated with impaired lymphatic vascular function as well as increased cardiac fibrosis and inflammation, whereas VEGFCc156s treatment ameliorated these pathologies. Interestingly, angiotensin II-infused mice treated with VEGFCc156s also demonstrated a delayed and dramatic response to reverse angiotensin II-induced hypertension. Thus, we propose that the beneficial effects of the lymphangiogenic therapy VEGFCc156s on angiotensin II infusion-induced cardiac dysfunction are multifaceted and mediated by its concomitant effects on cardiac lymphatic function, inflammatory and fibrotic gene expression, and systemic hypertension. Results Lymphatic endothelial markers associate with cardiac comorbidities and are reduced in human failing hearts To investigate the role of the lymphatic vasculature in human heart failure and cardiac dysfunction, we first analyzed human genome wide association studies (GWAS) for single-nucleotide polymorphisms (SNPs) that were near lymphatic marker gene loci and were associated with human cardiovascular or metabolic comorbidities. Interestingly, several SNPs near lymphatic endothelial markers VEGFC, LYVE1, FLT4 loci are associated with cardiovascular and metabolic phenotypes including cardiac hypertrophy in humans (Fung et al., 2019; Johansson et al., 2010; justice et al., 2019; Kettunen et al., 2016; Kichaev et al., 2019; Lotta et al., 2018; Lu et al., 2017; Nasa Sinnott-Armstrong et al., 2019; Saxena et al., 2010; van der Harst and Verweij, 2018; Wild et al., 2017; Xu et al., 2018; Table 1). In addition, human heart samples were obtained from healthy donors or patients with chronic heart failure, and the gene expression of lymphatic endothelial markers VEGFC, LYVE1, PDPN, and FLT4 was assessed. A significant (19.6% for VEGFC, 38.8% for LYVE1 and 21.7% for FLT4 on average) reduction was observed for lymphatic markers VEGFC, LYVE1, and FLT4, and a 31.6% reduction was observed in PDPN (p=0.07) in hearts from chronic heart failure patients compared with hearts from healthy donors (Figure 1A–D, Figure 1—source data 1), suggesting that the cardiac lymphatic vasculature is impaired in human chronic heart failure. Table 1 SNPs mapping to lymphatic endothelial markers associate with cardiac comorbidities. SNPs in human genetic loci, where lymphatic endothelial marker genes VEGFC, LYVE1, FLT4 are located, are associated with cardiovascular and metabolic phenotypes. MRVI1, murine retrovirus integration site one homolog. ADM, adrenomedullin. CTR9, RNA polymerase-associated protein CTR9 homolog. MGAT1, alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase. SCGB3A1, Secretoglobin Family 3A Member 1. OR2Y1, olfactory receptor family two subfamily Y member 1. SNPNearest gene(s)Associated phenotypep ValueReferencers2333496VEGFCWaist-to-hip ratio (WHR) adj BMI8.00E-11Lotta, et al.rs7660760VEGFCLeft ventricle wall thickness4.00E-07Wild, et al.rs309795VEGFC2 hr Glucose Adj. for BMI2.22E-06Saxena, et al.rs114108584VEGFCIdiopathic dilated cardiomyopathy9.00E-06Xu, et al.rs11042906LYVE1 and MRVI1Systolic BP2.00E-14Kichaev, et al.rs2218793LYVE1 and ADMHigh density lipoprotein cholesterol (HDL-C)1.60E-11Sinnott-Armstrong, et al.rs11042937LYVE1 and CTR9Coronary artery disease3.00E-10Van der harst, et al.rs7940646LYVE1 and MRVI1Triglycerides 2017 Mostly European and East Asian2.93E-08Lu, et al.rs10840457LYVE1 and MRVI1Arterial stiffness index3.00E-08Fung, et al.rs11603178LYVE1 and MRVI1Diastolic BP9.85E-08Nealelab-uk-biobankrs12807023LYVE1 and ADMPlasma + serum IDL-TAG levels8.21E-07Kettunen, et al.rs12807023LYVE1 and ADMPlasma + serum XS-VLDL values (concentration, TAG)5.41E-06Kettunen, et al.rs634501FLT4 and MGAT1High density lipoprotein cholesterol (HDL-C)9.35E-08Lu, et al.rs142342609FLT4 and SCGB3A1WHR adjusted BMI7.00E-07Justice, et al.rs12517906FLT4 and OR2Y1Body mass index (females)6.00E-06Johansson, et al. Figure 1 Download asset Open asset Lymphatic endothelial markers are reduced in human failing heart. (A–D) Expression of lymphatic endothelial marker genes VEGFC (A), LYVE1 (B), PDPN (C), FLT4 (D) in hearts from healthy human donors (HH) and hearts from patients with chronic heart failure (CHF). The data were normalized to the expression of the housekeeping gene HPRT1 and represented as fold change from the HH group (n = 16–18 for HH and n = 10–11 for CHF). Data are mean ± s.d. Student's t-test was used for statistics. *p<0.05. Figure 1—source data 1 Clinical information. https://cdn.elifesciences.org/articles/58376/elife-58376-fig1-data1-v2.xlsx Download elife-58376-fig1-data1-v2.xlsx Figure 1—source data 2 Raw data supporting Figure 1. https://cdn.elifesciences.org/articles/58376/elife-58376-fig1-data2-v2.xlsx Download elife-58376-fig1-data2-v2.xlsx VEGFCc156s prevented angiotensin II-induced cardiac dysfunction To examine the role of the lymphatic vasculature in cardiac dysfunction further, we used a model of angiotensin II infusion, which promotes cardiac dysfunction directly via its effects on cardiomyocytes (Mazzolai et al., 2000; Mehta and Griendling, 2007) and indirectly by its action on the renin-angiotensin system to induce hypertension (Mehta and Griendling, 2007). To induce lymphangiogenesis in this model, we used the lymphangiogenic growth factor mutant VEGFCc156s, which specifically activates vascular endothelial growth factor receptor 3 (VEGFR3) but not vascular endothelial growth factor receptor 2 (VEGFR2) in vitro (Joukov et al., 1998) and enhances lymphangiogenesis in vivo (Klotz et al., 2015). Pharmacokinetic analysis of single subcutaneous dosing of VEGFCc156s indicated that VEGFCc156s has a short half-life in plasma (Figure 2—figure supplement 1A). Thus, we hypothesized that continuous infusion to maintain a stable increase in plasma VEGFCc156s concentration would be the most effective mode of administration. Therefore, an osmotic pump was implanted subcutaneously to infuse angiotensin II and VEGFCc156s simultaneously (Figure 2A). The VEGFCc156s infusion was calculated to maintain a two-fold increase in plasma VEGFC concentration, and the analysis at endpoint confirmed a significant 1.9-fold average increase in plasma VEGFC concentration in the treated mice compared with control mice (Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset VEGFCC156S treatment prevented angiotensin II-induced cardiac dysfunction. (A) Experimental design for animal studies. Angiotensin II (angII) was infused to induce cardiac dysfunction, and VEGFCc156s (VEGFC) was infused as a lymphangiogenic therapy via the same subcutaneously-implanted osmotic pump. Bovine serum albumin (BSA) was loaded to the pumps for control (ctrl) and angII groups to balance for the loading of VEGFCc156s in the therapeutic arm. (B–E) Echocardiography was conducted 6 weeks after minipump implantation. Fractional Shortening (FS) (B), Ejection Fraction (EF) (C), Left Ventricular (LV) mass index (D) and Relative Wall Thickness (RWT) (E) are shown (n = 17–24/group). (F–G) Heart weight (F) and lung weight (G) (normalized to tibia length) of the animals at euthanasia (n = 7–18/group). (H) RNA was isolated from the mouse hearts, and quantitative RT-PCR was performed for Nppb and normalized to the expression of housekeeping gene Hprt. The data were normalized to ctrl group and represented as fold change (n = 8–9/group). (I) Mouse hearts were fixed, OCT embedded, sectioned, and stained for podoplanin, smooth muscle actin and DAPI (light blue). Representative images of podoplanin-positive lymphatic vessels (green) and smooth muscle actin-positive arteries (red) for each group were shown. Scale bar, 200 μm. (J) Quantification of podoplanin-positive lymphatic vessel density (J, normalized to total area and represented as fold change to ctrl group) and podoplanin-positive lymphatic vessel lumen area (K) in mouse hearts (n = 8–10 animal/group, n = 10–12 sections/animal). Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for all figures except 2J and 2K (a linear mixed model was used for statistics). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant. Figure 2—source data 1 Raw data supporting Figure 2. https://cdn.elifesciences.org/articles/58376/elife-58376-fig2-data1-v2.xlsx Download elife-58376-fig2-data1-v2.xlsx Echocardiography was conducted at endpoint to examine cardiac function, and the results indicated that angiotensin II infusion induced cardiac hypertrophy and impaired cardiac contractility. In contrast, VEGFCc156s treatment prevented angiotensin II-induced impairments in cardiac function (Figure 2B–E, Figure 2—figure supplement 1C–1G). Consistent with the echocardiography results, angiotensin II infusion also significantly increased heart weight and lung weight, which possibly reflects an increase in lung edema (King and Goldstein, 2020), whereas VEGFCc156s prevented the angiotensin II-induced increases in heart and lung weights (Figure 2F and G). Moreover, gene expression analysis revealed a significant increase in the expression of Nppb, encoding heart failure marker B-type natriuretic peptide (BNP), in angiotensin II-treated mouse hearts compared with control hearts, while VEGFCc156s significantly decreased Nppb expression in angiotensin II-infused hearts (Figure 2H). Finally, cardiomyocyte cell size was examined using wheat germ agglutinin (WGA), which stains the cell membrane. Whereas angiotensin II infusion significantly increased cardiomyocyte size and heterogeneity, VEGFCc156s treatment prevented the change in cell size heterogeneity in angiotensin II-infused mouse hearts (Figure 2—figure supplement 1H–1J), confirming the beneficial effects of VEGFCc156s on angiotensin II-induced cardiac hypertrophy. To understand how VEGFCc156s ameliorated cardiac dysfunction, the effect of VEGFCc156s on cardiac lymphatic vascular marker expression and VEGFR3-mediated signaling cascades was examined. Similar to what was observed in human hearts (Figure 1), a significant reduction in lymphatic endothelial marker podoplanin expression was observed in angiotensin II-infused hearts compared with control hearts, indicating a potential reduction in cardiac lymphatic vascular density. Interestingly, VEGFCc156s treatment restored podoplanin expression in angiotensin II-infused hearts back to control level, indicating that VEGFCc156s treatment may prevent the loss of cardiac lymphatic vessels during cardiac dysfunction (Figure 2—figure supplement 1K and L). Consistent with the protein expression analysis, immunohistochemistry revealed a significant reduction in podoplanin-positive lymphatic vessel density in angiotensin II-infused hearts, which was restored by VEGFCc156s treatment (Figure 2I and J). More interestingly, podoplanin-positive lymphatic vessel lumen area was significantly increased in the hearts of VEGFCc156s + angiotensin II-infused animals compared with animals that had been treated with angiotensin II alone, indicating that VEGFCc156s may improve lymphatic transport function in diseased animals (Figure 2K). Consistent with the ability of VEGFC to activate Akt and Erk pathways, we observed increased phospho-Akt levels and a trend towards an increase in phospho-Erk in VEGFCc156s-treated hearts compared with control or angiotensin II-infused hearts, indicating the activation of signaling pathways that promote survival and growth by VEGFCc156s treatment (Figure 2—figure supplement 1K, M and N). To further characterize the systemic lymphangiogenic effect of VEGFCc156s, we examined lymphatic vessel diameter and density in the ear skin by immunohistochemistry. Similar to its effects on cardiac lymphatic vessels, VEGFCc156s treatment also significantly increased lymphatic vessel diameter and lymphatic vascular density in the ear skin of treated animals, supporting a systemic lymphangiogenic response induced by VEGFCc156s treatment (Figure 2—figure supplement 1O–1R). VEGFCc156s improved cardiac lymphatic vascular function To investigate whether the changes in podoplanin-positive lymphatic vessel density and lumen area in heart were translated into functional changes, we conducted lymphangiography to directly evaluate cardiac lymphatic transport function. FITC-dextran and cadaverine were injected intravenously to label the cardiac blood vasculature and reveal any vascular leak, respectively. Next, fluorescent qdots were injected into the apex of the heart. Qdots are macromolecules that diffuse into interstitial fluid and may be cleared by the lymphatic vasculature and transported back to the blood circulation (Henri et al., 2016; Shimizu et al., 2018). Angiotensin II treatment induced vascular leakage, leading to interstitial accumulation of cadaverine in angiotensin II-infused hearts. In contrast, VEGFCc156s-treated, angiotensin II-infused animals showed significantly reduced cadaverine accumulation in heart, indicating reduced leakage and improved fluid clearance (Figure 3A and B, Figure 3—figure supplement 1). We observed that in all animals, the qdots were transported back to the blood circulation within 5 mins after injection. However, quantification of the plasma qdot concentration, which is an indirect parameter to evaluate cardiac lymphatic transport efficiency, demonstrated a significant decrease in angiotensin II-infused mice compared with control mice, indicating impaired cardiac lymphatic transport. Conversely, VEGFCc156s treatment in the angiotensin II-infused mice significantly increased the plasma qdot concentration compared with the angiotensin II-infused mice, indicating improved cardiac lymphatic transport (Figure 3C). Concomitantly, a trend toward an increased heart wet-to-dry weight ratio was observed in angiotensin II-infused hearts, which was significantly reduced with VEGFCc156s treatment, indicating improved regulation of cardiac fluid balance in VEGFCc156s-treated mice (Figure 3D). These results demonstrated that VEGFCc156s treatment improved cardiac lymphatic vascular function in angiotensin II-infused animals, which may facilitate fluid balance regulation and contribute to the improved cardiac function observed after VEGFCc156s treatment. Figure 3 with 1 supplement see all Download asset Open asset VEGFCC156S treatment improved cardiac lymphatic vascular function. (A) Representative lymphangiography images of adult mouse hearts. FITC-dextran (green) indicates blood vessels, cadaverine (orange) indicates vascular leak and qdot (red) indicates injection site and cardiac lymphatics. Objective, 1x. Scale bar, 2000 μm. (B) Analysis of cadaverine-positive area (normalized to total area) in heart 5 mins after injection. The data were normalized to ctrl group and represented as fold change (n = 10–19/group). (C) Analysis of qdot plasma concentration 5 mins after injection (n = 10–16/group). (D) Wet-to-dry weight ratio of hearts (n = 9–22/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for statistics. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant. Figure 3—source data 1 Raw data supporting Figure 3. https://cdn.elifesciences.org/articles/58376/elife-58376-fig3-data1-v2.xlsx Download elife-58376-fig3-data1-v2.xlsx VEGFCc156s ameliorated angiotensin II-induced inflammatory responses To further characterize the effects of VEGFCc156s on angiotensin II infusion-induced cardiac dysfunction, we conducted RNA sequencing (RNA-seq) analysis to profile the transcriptional changes in different cell populations from the heart. Transcriptional changes in diaphragm lymphatic endothelial cells (LECs) were also profiled to evaluate the effect of VEGFCc156s on peripheral lymphatics, as the treatment is systemic. To efficiently distinguish LECs from blood endothelial cells (BECs), we generated a Prox1-eGFP knock-in mouse model in which eGFP expression is driven by the endogenous promoter of the lymphatic endothelial marker Prox1 gene using CRISPR/Cas9 technology and confirmed the eGFP signals from lymphatic vessels in the heart and the diaphragm (Figure 4—figure supplement 1A–1C). The Prox1-eGFP mice were next used for in vivo studies following the study design shown in Figure 2A. From the animals, LECs, BECs and cardiomyocytes were isolated from the heart, and LECs were additionally isolated from the diaphragm following an optimized protocol (Figure 4—figure supplement 1D) and used for RNA-seq. A principal component analysis of the normalized expression counts across cell types indicated that the samples clustered based on cell population and showed a distinct expression profile (Figure 4—figure supplement 1E). Differential expression and signaling pathway analysis revealed that angiotensin II significantly altered gene expression in all four cell populations. VEGFCc156s treatment reversed many of these changes in heart and diaphragm LECs but not in cardiomyocytes or cardiac BECs, confirming the specificity of VEGFCc156s effects on the lymphatic vasculature (Figure 4A–C, Figure 4—figure supplement 1F–1H). A causal reasoning analysis was performed to predict potential upstream regulators for the modulated genes. This analysis predicted that angiotensin II is a causal factor for the gene expression changes in cardiomyocytes from both the angiotensin II-infused and the VEGFCc156s-treated, angiotensin II-infused group, confirming similar effects of angiotensin II on cardiomyocytes at endpoint (Figure 4—figure supplement 1I). One common causal factor and related pathway that was activated by VEGFCc156s treatment in LECs from both heart and diaphragm is MYCN, suggesting the activation of lymphatic endothelial cell growth and proliferation by VEGFCc156s (Figure 4—figure supplement 1J). Notably, angiotensin II infusion upregulated the expression of multiple genes involved in inflammation, chemotaxis and related pathways in LECs from both heart and diaphragm. Conversely, VEGFCc156s treatment downregulated the expression of these inflammatory and chemotactic genes (Figure 4A–C). Causal reasoning analysis also suggested that toll-like receptor 4 (TLR4) and toll-like receptor 3 (TLR3) signaling pathways were activated in LECs from angiotensin II-infused hearts but were reduced in LECs from VEGFCc156s-treated, angiotensin II-infused hearts, supporting a beneficial effect of VEGFCc156s treatment on angiotensin II infusion-induced inflammatory pathways in LECs (Figure 4—figure supplement 1J). Figure 4 with 1 supplement see all Download asset Open asset VEGFCC156S treatment ameliorated angiotensin II-induced inflammatory responses. (A–B) Heat map visualization of all FDR-significant differential expression changes in genes comparing angII and ctrl groups (angII vs ctrl), angII+VEGFC and angII groups (angII+VEGFC vs angII), and angII+VEGFC and ctrl groups (angII+VEGFC vs ctrl) in heart lymphatic endothelial cells (LECs) and diaphragm LECs. The differential expression test-statistics are indicated by the shading in the heatmap and several genes that are significantly regulated by VEGFC treatment are shown. (C) Top 15 affected signaling pathways in angII+VEGFC vs angII comparison for heart LECs and diaphragm LECs from gene ontology (GO) pathway analysis. (D) KC/GRO levels in mouse plasma (n = 16–21/group). (E–F) IP-10 and MDC levels in native mouse skin lysates. The values of IP-10 levels were log-transformed, and the values were normalized to the ctrl group and represented as fold change (n = 7–9/group). (G) Mouse hearts were fixed, OCT embedded, sectioned, and stained for CD68 (green), smooth muscle actin (red) and DAPI (light blue). Representative images for each group are shown and CD68/DAPI double-positive macrophages are indicated with arrowheads. Scale bar, 100 μm. (H–I) CD68/DAPI double-positive macrophage density and macrophage density near smooth muscle actin-positive arteries (normalized to total area and represented as fold change to ctrl group) in mouse hearts (n = 8–9/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for all figures except 4E (One-way ANOVA with Tukey's posthoc was used for log-transformed data). *p<0.05, **p<0.01, n.s. not significant. Figure 4—source data 1 Raw data supporting Figure 4. https://cdn.elifesciences.org/articles/58376/elife-58376-fig4-data1-v2.xlsx Download elife-58376-fig4-data1-v2.xlsx To validate the findings from the RNA-seq analysis, we examined circulating chemokine levels as well as chemokine levels in skin, which is highly vascularized with lymphatic capillaries. Consistent with the RNA-seq analysis, a significant 43% increase in plasma KC/GRO levels was noted in angiotensin II-infused mice compared with control mice, and VEGFCc156s treatment trended to reduce plasma KC/GRO levels in angiotensin II-infused mice (Figure 4D). Furthermore, we observed significantly increased levels of several chemokines (37.2% for chemokine interferon γ-induced protein 10 (IP-10), 68% for macrophage-derived chemokine (MDC) and 30% for KC/GRO on average) in skin of angiotensin II-infused mice compared with control mice, while VEGFCc156s treatment significantly reduced the levels or induced a trend toward a reduction in these chemokines (Figure 4E and F, Figure 4—figure supplement 1K). In addition, angiotensin II infusion significantly increased general macrophage density in heart as well as macrophage density near arteries as assessed by CD68 immunostaining, indicating increased local macrophage content, whereas VEGFCc156s treatment significantly reduced these parameters (Figure 4G–I). Altogether, the results suggest that VEGFCc156s treatment ameliorated angiotensin II infusion-induced inflammatory and chemotactic responses potentially by affecting the LEC transcriptome, which may contribute to the efficacy observed in the disease model. VEGFCc156s ameliorated angiotensin II-induced hypertension Immune cells and inflammatory processes can play a role in the pathogenesis of hypertension (Caillon et al., 2019), and angiotensin II-induced hypertension promotes cardiac dysfunction. Therefore, the effect of VEGFCc156s treatment on angiotensin II-induced hypertension was examined by conducting a telemetry study as detailed in Figure 5A. Telemetry recordings started 3 days before minipump implantation and lasted for 5 weeks after surgery. The time-course analysis of mean blood pressure demonstrated a significant increase in blood pressure and the development of hypertension in angiotensin II-infused mice compared with control mice. Interestingly, VEGFCc156s-treated, angiotensin II-infused mice showed a similar significant increase in both systolic and diastolic blood pressure as those that were treated with angiotensin II alone in the first However, at week the VEGFCc156s-treated angiotensin II group demonstrated a in systolic and diastolic blood pressure that was to control mice by week 4 (Figure Figure supplement 1). To examine whether VEGFCc156s angiotensin II aortic to reduce blood was assessed to examine aortic and both angiotensin II-infused mice and VEGFCc156s-treated, angiotensin II-infused mice showed a significant increase in compared with control mice, indicating that VEGFCc156s treatment not angiotensin II aortic stiffness and (Figure In addition, the expression of which the for angiotensin was induced in both angiotensin II-infused mice and VEGFCc156s-treated, angiotensin II-infused mice to a similar compared with the from control mice, indicating a similar response in to with continuous angiotensin II infusion (Figure These results suggest that VEGFCc156s treatment ameliorated angiotensin II-induced hypertension angiotensin II aortic stiffness and the underlying the beneficial effects of VEGFCc156s on angiotensin II-induced cardiac dysfunction. Figure 5 with 1 supplement see all Download asset Open asset VEGFCC156S treatment ameliorated angiotensin II-induced hypertension. (A) Experimental design for telemetry (B) blood pressure of angII and mice after minipump implantation (a linear mixed model was used for statistics). Analysis of mean blood pressure at (C), week 1 (D) and week 4 (E) (n = (F) weeks after minipump implantation (n = (G) RNA was from mouse and was performed for and normalized to Hprt. The data were normalized to the ctrl group and represented as fold change (n = 16–21/group). Ctrl, control. AngII, angiotensin II. VEGFC, VEGFCc156s. Data are mean ± s.d. One-way ANOVA with Bonferroni posthoc was used for statistics. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. not significant. Figure data 1 Raw data supporting Figure Download VEGFCc156s improved angiotensin II-induced dysfunction To validate the effect of VEGFCc156s treatment on angiotensin II infusion-induced angiotensin II-induced changes in were on the of and and stained by a angiotensin II infusion induced vascular hypertrophy in the of both angiotensin II-infused and VEGFCc156s-treated angiotensin II-infused animals, which is with the action of angiotensin II as a (Mehta and Griendling, 2007). However, angiotensin II-infused VEGFCc156s-treated animals showed a reduced of angiotensin II-induced as interstitial cell and in the compared with angiotensin II-infused animals, indicating that VEGFCc156s treatment angiotensin II infusion-induced injury (Figure supplement and In addition, demonstrated increased