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Direct evidence of VEGF-mediated neuroregulation and afferent explanation of blood pressure dysregulation during angiogenic therapy
doi: 10.2478/fzm-2021-0015
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Abstract:
Objective Oncocardiology is increasingly hot research field/topic in the clinical management of cancer with anti-angiogenic therapy of vascular endothelial growth factor (VEGF) that may cause cardiovascular toxicity, such as hypertension via vascular dysfunction and attenuation of eNOS/NO signaling in the baroreflex afferent pathway. The aim of the current study was to evaluate the potential roles of VEGF/VEGF receptors (VEGFRs) expressed in the baroreflex afferent pathway in autonomic control of blood pressure (BP) regulation. Methods The distribution and expression of VEGF/VEGFRs were detected in the nodose ganglia (NG) and nucleus of tractus solitary (NTS) using immunostaining and molecular approaches. The direct role of VEGF was tested by NG microinjection under physiological and hypertensive conditions. Results Immunostaining data showed that either VEGF or VEGFR2/VEGFR3 was clearly detected in the NG and NTS of adult male rats. Microinjection of VEGF directly into the NG reduced the mean blood pressure (MBP) dose-dependently, which was less dramatic in renovascular hypertension (RVH) rats, suggesting the VEGF-mediated depressor response by direct activation of the 1st-order baroreceptor neurons in the NG under both normal and disease conditions. Notably, this reduced depressor response in RVH rats was directly caused by the downregulation of VEGFR2, which compensated the up regulation of VEGF/VEGFR3 in the NG during the development of hypertension. Conclusion It demonstrated for the first time that the BP-lowering property of VEGF/VEGFRs signaling via the activation of baroreflex afferent function may be a common target/pathway leading to BP dysregulation in anti-angiogenic therapy. -
Figure 1. Immunohistochemical characterization of the expression and distribution of VEGF, VEGFR2 and VEGFR3 in the NG and NTS.
(A) The results of conventional immunohistochemical experiments carried out with specific antibodies against DAPI (blue), HCN1 (red), and VEGF, VEGFR2 or VEGFR3 (green) to detect the cellular/subcellular distributions of VEGF, VEGFR2 and VEGFR3 in the NG of adult male SD rats. Yellow and white arrowheads represent HCN1-positive (myelinated A-type afferents) and HCN1-negative (unmyelinated C-type afferents) neurons, respectively. Scale bar = 50 μm. (B) The representative immunostaining images of whole brainstem section showing the distribution of VEGF, VEGFR2 and VEGFR3 in the NTS. Sol: solitary tract; the scale bar = 2.0 mm. SolL: nucleus of the solitary tract (Sol); SolVL: -ventrolateral part; SolV: -ventral part; SolM: -medial part; SolDM: -dorsomedial part; SolIM: -intermediate part. The fluorescent bar represents the expression level. The negative staining as a control for both NG and NTS is presented in supplemental materials.
Figure 2. VEGF-mediated BP reduction in control and RVH rats following direct microinjection of VEGF into the NG.
(A and B) Changes in BP and body weight of Ctrl and RVH rats during 4 weeks after surgery; (C) Representative recordings of VEGF-medicated BP reduction following administration of a series of doses of VEGF via direct microinjection into the left side of the NG in control (Ctrl) and RVH model rats. Dotted lines represent the time points of microinjections; (D) Summarized data of VEGF-mediated BP reduction with different doses of VEGF; (E) Summarized data showing the recovery time of VEGF-mediated BP reduction. The data are expressed as mean ± SD and n = 5 rats for each group, *P < 0.05 and **P < 0.01 vs. Ctrl.
Figure 3. Changes in cardiac function, blood pressure, and baroreflex sensitivity in RVH rats.
(A) Cardiac parameters determined by echocardiograph in control and RVH rats. Data are presented as mean ± SD, n = 4, *P < 0.05 vs. control (Ctrl). PSLAX: parasternal long axis; LVIDs/LVIDd: systolic/diastolic left ventricular internal diameter; IVSs/IVSd: systolic/diastolic left ventricular septum thickness; LVPWs/LVPWd: systolic/diastolic left ventricular posterior wall; EF: ejection fraction; FS: fractional shortening. (B) Representative recordings of mean arterial blood pressure (MAP, purple) and heart rate (blue) collected from Ctrl (n = 5) and RVH (n = 5) rats treated with 1, 3, and 10 μg/kg of SNP and PE, respectively. Scale bars are applied to all recordings. (C-D) The summarized changes of BRS (ΔHR/ΔMABP, bpm/mmHg) after treatment with SNP or PE at different concentrations. Data are expressed as means ± SD. *P < 0.05 and **P < 0.01 vs. Ctrl.
Figure 4. Relative mRNA and protein expression levels of VEGF, VEGFR2 and VEGFR3 in the NG and NTS.
(A-C) Relative mRNA and protein expression levels (fold change) and representative protein bends of VEGF, VEGFR2, and VEGFR3 in the NG of control and RVH model rats compared with internal control (GAPDH); (D-F) Relative mRNA and protein expression levels (fold change) and representative protein bends of VEGF, VEGFR2, and VEGFR3 in the NTS of control and RVH model rats compared with internal control (GAPDH). Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 vs. Ctrl, n = 4 duplicated tests and tissue specimens collected from 6 rats for both PCR and Western blot analyses.
Figure 5. The expression alterations of VEGF, VEGFR2 and VEGFR3 in the kidney.
(A) Expression alterations (WB and PCR) of VEGF in the kidney of control and RVH rats compared with the internal control (GAPDH). Data are presented as mean ± SD; *P < 0.05 vs. control, n = 4; (B) Expression changes (WB and PCR) of VEGFR2 in the kidney of control and RVH rats compared with the internal control (GAPDH). Data are presented as mean ± SD; *P < 0.05 vs. control, n = 4; (C) Expression alterations (WB and PCR) of VEGFR3 in the kidney of control and RVH rats compared with the internal control (GAPDH). Data are presented as mean ± SD; *P < 0.05 vs. control, n = 4.
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