| Objective:Long term high salt intake is an important environmental factor involved in the aggravation of hypertension and related cardiovascular disease. High salt loading can not only induce hypertension but detrimentally influence target organ independently of its effects on blood pressure. Recent studies have shown that TonEBP/VEGF-C/VEGFR-3signaling pathway in mononuclear phagocyte system (MPS) cells of skin interstitium provides a buffering mechanism for blood pressure homeostasis. Moreover, blocking this pathway by MPS cell depletion can induce salt-sensitive hypertension. Our previous study has demonstrated that chronic high salt intake (8%NaCl) for12weeks can lead to a markedly MPS cell infiltration and lymphangiogenesis in left ventricule (LV) of spontaneously hypertensive rats (SHR), which indicates upregulation of TonEBP/VEGF-C/VEGFR-3signaling pathway in heart after high salt intake may be existed. But its pathophysiological role in process of hypertensive LV remodeling is still unclear. The present work was designed to investigate the dynamics of lymphangiogenesis and MPS cell infiltration in heart of high salt loaded SHR in different time points and to demonstrate the role of VEGF-C/VEGFR-3signaling pathway in high salt intake induced hypertensive LV remodeling.Methods:(1)7-week-old male SHR were divided into low salt group (LS,0.5%NaC1) and high salt group (HS,8%NaC1), and each group were then divided into8-week,12-week and16-week subgroups.12-week and16-week groups were anesthetized for hemodynamic measurements before sarcrifice. At the end of follow-ups of each subgroup, rat hearts were dissected for pathological analysis and homogenized for measuring expression levels of related genes and proteins.(2) We constructed pLPCX-△N△C/VEGF-C/Cys152Ser (pLPCX-VC, overactivating VEGFR-3) and pLPCX plasmids using pSecTag-△N△C/VEGF-C/Cys152Ser (pSecTag-VC) and pLPCX-VEGFR-3-Rg (pLPCX-VRg, a trap of VEGF-C). After verified by sequencing, PCR and enzyme digestion, the three retroviral plasmids were transfected into PT67cells and positive clones were collected for retroviral production. The retroviral supernatants were collected and concentrated for further study. The expressions of retroviruses in RAW264.7cells and rats were measured by Western blot analysis.(3)7-week-old male SHR were divided into LS group and HS group, same age Wistar Kyoto rats (WKY) were given a LS diet for negative control. HS rats were then divided into four subgroups:HS group, HS+pLPCX-VC retrovirus (HS+VC) group, HS+pLPCX-VRg retrovirus (HS+VRg) group and HS+pLPCX retrovirus (HS+Ctr) group for administering different kinds of retroviruses via tail vein after high salt intake for8weeks. All rats were anesthetized for echocardiography and hemodynamic measurements before sarcrifice. Then hearts were prepared for pathology analysis and homogenized for determining expression levels of related genes and proteins.Results:(1) Before12-week, systolic blood pressure (SBP) of HS group gradually increased, then decreased. LV mass index (LVMI) was significantly higher at each time points compared with LS group ([<0.05) while the body weight was markedly decreased (p<0.05). Hemodynamic analysis showed systolic and diastolic function were markedly decreased in16-week HS group (p＜0.05). The mRNA levels of Prox-1and TonEBP were both significantly increased in8-week HS group while no markedly differences were found in Lyve-1, podoplanin and VEGF-C. The mRNA levels of lymphatic cell markers (Prox-1, Lyve-1and podoplanin), TonEBP and VEGF-C were all significantly increased in12-week and16-week HS groups (p<0.05). Protein levels of VEGF-C and Prox-1were also markedly increased after high salt intake for12weeks (p＜0.05). Meanwhile, TonEBP protein level of12-week HS group was also significantly increased (p<0.05). Pathological analysis showed cross-sectional area of cardiomyocytes, myocardial interstitium and perivascular collogen deposition, number of MPS cell infiltration, and number of blood and lymphatic vessels were all significantly increased in12-week HS group (all p<0.05).(2) pLPCX-VC and pLPCX plasmids were successfully constructed and verified. The three retroviruses were packaged out after transfecting PT67cells and puromycin selection. The retroviral supernatants were collected and concentrated. Retroviral titers were:pLPCX-VC retrovirus (RetroVC)1.2x108CFU/mL, pLPCX-VRg retrovirus (RetroVRg)2.84×108CFU/mL and pLPCX retrovirus (RetroCtr)3.07×108 CFU/mL. Western blot analysis showed RetroVC and RetroVRg were successfully expressed in RAW264.7cells and rats.(3) Body mass was markedly decreased while LV mass and LVMI were both significantly increased in HS groups compared with WKY and LS groups (p<0.05). The blood pressure of HS+VC group was significantly decreased while HS+VRg group was notably increased compared with HS and HS+Ctr group (p<0.05). Echocardiography analysis demonstrated LV enlargement was significantly ameliorated in HS+VC group compared with HS+VRg group (p<0.05), contractility and LV function were also partly reserved (p<0.05). Hemodynamic analysis showed left ventricular end diastolic pressure (LVEDP) was significantly decreased in HS+VC group compared with HS+VRg group (p<0.05), while-dP/dlmin(active diastolic fuction) and+dP/dtmax (systolic function) were both significantly improved in HS+VC group compared with other HS groups (p<0.05).The mRNA levels of lymphatic markers were all significantly increased in HS+VC group while markedly inhibited in HS+VRg group (p＜0.05). TonEBP mRNA level was significantly increased in HS groups (p＜0.05). VEGF-C mRNA level was significantly higher in HS+VC group compared with other HS groups (p＜0.05). Prox-1protein level was also markedly increased in HS+VC group (p＜0.05). Accompanied with lymphangiogenesis, RetroVC treatment significantly ameliorated cardiomyocyte hypertrophy, decreased perivacular and myocardial interstitial collogen deposition and alleviated MPS cell infiltration(p＜0.05). In contrast, RetroVRg treatment resulted an opposite effect on LV remodeling.Conclusions:The present study demonstrates that chronic high salt intake contributes to myocardial intistitial MPS cell-infiltration and lymphangiogenesis in SHR. Upregulation of VEGF-C mediated lymphangiogenesis could attenuate myocardial intistitial MPS cells infiltration, reserve LV systolic and diastolic function and improve hypertensive LV remodeling. Our findings highlight VEGF-C as a promising therapeutic target to improve hypertensive LV remodeling induced by long-term high salt intake. |
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