Chengwen Sun

The blood–brain barrier (BBB) restricts the entry of therapeutic agents into the brain cardiovascular regulatory region, potentially contributing to drug-resistant hypertension. Objective: The objective of this study was to overcome this limitation by modifying PEGylated liposomes with transferrin (Tf) to facilitate Tf receptor binding at the BBB and penetratin (Pen), a cell-penetrating peptide, to enhance neuronal uptake. Methods: This study evaluated the efficacy of Tf-Pen-liposomes in delivering angiotensin-converting enzyme 2 (ACE2) or EGFP (control) genes across the BBB in rats. In addition, the therapeutic effect of intravenous administration of Tf-Pen-Lip carrying plasmid DNA encoding ACE2 (Tf-Pen-Lip-pACE2) was tested in a neurogenic hypertension model induced by intracerebroventricular (ICV) infusion of angiotensin II (Ang II) via osmotic pump implantation and brain cannulation. Results: Conjugation with Tf and Pen significantly enhanced liposome-mediated gene transfection in cultured cells and increased transport across an in vitro BBB model. In vivo, intravenous administration of Tf-Pen-Lip-pACE2 or Tf-Pen-Lip-pGFP successfully elevated ACE2 or EGFP expression, respectively, in the hypothalamic paraventricular nucleus (PVN). Chronic ICV infusion of Ang II produced a sustained increase in blood pressure and heart rate, accompanied by sympathetic overactivation and elevated arginine vasopressin (AVP) secretion, hallmarks of neurogenic hypertension. Notably, intravenous Tf-Pen-Lip-pACE2 treatment dramatically attenuated Ang II–induced neurogenic hypertension, whereas Tf-Pen-Lip-pGFP had no effect on pressor responses, sympathetic activity, or AVP secretion. Conclusions: This dual-functionalized liposomal delivery system effectively transported the ACE2 gene across the BBB into the brain, increased ACE2 expression, and markedly attenuated neurogenic hypertension following systemic administration.

Mechanisms by which BK_Ca (large-conductance calcium-sensitive potassium) channels are involved in vascular remodeling in hypertension are not fully understood. Vascular smooth muscle cell (VSMC) proliferation and vascular morphology were compared between hypertensive and normotensive rats. BK_Ca channel activity, protein expression, and interaction with IP3R (inositol 1,4,5-trisphosphate receptor) were examined using patch clamp, Western blot analysis, and coimmunoprecipitation. On inside-out patches of VSMCs, the Ca²⁺-sensitivity and voltage-dependence of BK_Ca channels were similar between hypertensive and normotensive rats. In whole-cell patch clamp configuration, treatment of cells with the IP3R agonist, Adenophostin A (AdA), significantly increased BK_Ca channel currents in VSMCs of both strains of rats, suggesting IP3R-BK_Ca coupling; however, the AdA-induced increases in BK_Ca currents were attenuated in VSMCs of hypertensive rats, indicating possible IP3R-BK_Ca decoupling, causing BK_Ca dysfunction. Co-immunoprecipitation and Western blot analysis demonstrated that BK_Ca and IP3R proteins were associated together in VSMCs; however, the association of BK_Ca and IP3R proteins was dramatically reduced in VSMCs of hypertensive rats. Genetic disruption of IP3R-BK_Ca coupling using junctophilin-2 shRNA dramatically augmented Ang II-induced proliferation in VSMCs of normotensive rats. Subcutaneous infusion of NS1619, a BK_Ca opener, to reverse BK_Ca dysfunction caused by IP3R-BK_Ca decoupling significantly attenuated vascular hypertrophy in hypertensive rats. In summary, the data from this study demonstrate that loss of IP3R-BK_Ca coupling in VSMCs induces BK_Ca channel dysfunction, enhances VSMC proliferation, and thus, may contribute to vascular hypertrophy in hypertension.

Apelin, by stimulation of APJ receptors, induces transient blood pressure (BP) reduction and positive inotropic effects. APJ receptors share high homology with the Ang II type 1 receptor; thus, apelin was proposed to play a protective role in cardiovascular disease by antagonizing the actions of Ang II. In this regard, apelin and apelin-mimetics are currently being studied in clinical trials. However, the chronic effect of apelin in cardiovascular regulation has not been fully investigated. In the current study, blood pressure (BP) and heart rate (HR) were recorded using a telemetry implantation approach in conscious rats, before and during chronic subcutaneous infusion of apelin-13, using osmotic minipumps. At the end of the recording, the cardiac myocyte morphology was examined using H&E staining, and cardiac fibrosis was evaluated by Sirius Red in each group of rats. The results demonstrated that the chronic infusion of apelin-13 did not change either BP or HR. However, under the same condition, the chronic infusion of Ang II induced significant BP elevation, cardiac hypertrophy, and fibrosis. Co-administration of apelin-13 did not significantly alter the Ang II-induced elevation in BP, changes in cardiac morphology, and fibrosis. Taken together, our experiments showed an unexpected result indicating that the chronic administration of apelin-13 did not alter basal BP, nor did it change Ang II-induced hypertension and cardiac hypertrophy. The findings suggest that an APJ receptor biased agonist could be a better therapeutic alternative for treatment of hypertension.

Hypertension is a major health concern globally. Elevated blood pressure, initiated and maintained by the brain, is defined as neurogenic hypertension (NH), which accounts for nearly half of all hypertension cases. A significant increase in angiotensin II-mediated sympathetic nervous system activity within the brain is known to be the key driving force behind NH. Blood pressure control in NH has been demonstrated through intracerebrovascular injection of agents that reduce the sympathetic influence on cardiac functions. However, traditional antihypertensive agents lack effective brain permeation, making NH management extremely challenging. Therefore, developing strategies that allow brain-targeted delivery of antihypertensives at the therapeutic level is crucial. Targeting nanotherapeutics have become popular in delivering therapeutics to hard-to-reach regions of the body, including the brain. Despite the frequent use of nanotherapeutics in other pathological conditions such as cancer, their use in hypertension has received very little attention. This review discusses the underlying pathophysiology and current management strategies for NH, as well as the potential role of targeted therapeutics in improving current treatment strategies.

Apelin-APJ receptor signaling regulates vascular tone in cerebral and peripheral arteries. We recently reported that apelin inhibits BK_Ca channel function in cerebral arteries, resulting in impaired endothelium-dependent relaxations. In contrast, apelin causes endothelium-dependent relaxation of coronary arteries. However, the effects of apelin on BK_Ca channel function in coronary arterial myocytes have not yet been explored. We hypothesized that apelin-APJ receptor signaling does not have an inhibitory effect on coronary arterial BK_Ca channels and hence does not alter nitric oxide (NO)-dependent relaxation of coronary arteries. Patch clamp recording was used to measure whole cell K⁺ currents in freshly isolated coronary smooth muscle cells. Apelin had no effect on the increases in current density in response to membrane depolarization or to NS1619 (a BK_Ca channel opener). Moreover, apelin did not inhibit NO/cGMP-dependent relaxations that required activation of BK_Ca channels in isolated coronary arteries. Apelin-APJ receptor signaling caused a marked increase in intracellular Ca²⁺ levels in coronary arterial smooth muscle cells, but failed to activate PI3-kinase to increase phosphorylation of Akt protein. Collectively, these data provide mechanistic evidence that apelin has no inhibitory effects on BK_Ca channel function in coronary arteries. The lack of inhibitory effect on BK_Ca channels makes it unlikely that activation of APJ receptors in coronary arteries would adversely affect coronary flow by creating a vasoconstrictive environment. It can be expected that apelin or other APJ receptor agonists in development will not interfere with the vasodilator effects of endogenous BK_Ca channel openers.

Cardiac hypertrophy is an adaptive response to cardiac overload initially but turns into a decompensated condition chronically, leading to heart failure and sudden cardiac death. The molecular mechanisms involved in cardiac hypertrophy and the signaling pathways that contribute to the switch from compensation to decompensation are not fully clear. The aim of the current study was to examine the role of PI3-kinases Class I (PI3KC1) and Class III (PI3KC3) in angiotensin (Ang) II-induced cardiac hypertrophy. The results demonstrate that treatment of cardiomyocytes with Ang II caused dose-dependent increases in autophagy, with an increasing phase followed by a decreasing phase. Ang II-induced autophagic increases were potentiated by inhibition of PI3KC1 with LY294002, but were impaired by inhibition of PI3KC3 with 3-methyladenine (3-MA). In addition, blockade of PI3KC1 significantly attenuated Ang II-induced ROS production and cardiomyocyte hypertrophy. In contrast, blockade of PI3KC3 potentiated Ang II-induced ROS production and cardiac hypertrophy. Moreover, blockade of PI3KC1 by overexpression of dominant negative p85 subunit of PI3KC1 significantly attenuated Ang II-induced cardiac hypertrophy in normotensive rats. Taken together, these results demonstrate that both PI3KC1 and PI3KC3 are involved in Ang II-induced cardiac hypertrophy by different mechanisms. Activation of PI3KC1 impairs autophagy activity, leading to accumulation of mitochondrial ROS, and, hence, cardiac hypertrophy. In contrast, activation of PI3KC3 improves autophagy activity, thereby reducing mitochondrial ROS and leads to a protective effect on Ang II-induced cardiac hypertrophy.

Passive exposure to second hand smoke is a risk factor for cardiovascular disorders, including coronary artery disease. Mechanisms underlying adverse effects of cigarette smoke on coronary arteries are poorly understood. Apelin is an endogenous ligand for APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. APJ receptors signal via G‐protein‐dependent and ‐independent pathways, including activation of G‐protein‐coupled‐receptor kinase 2 (GRK2). We previously reported that apelin causes relaxation of coronary arteries via the release of endothelium‐derived nitric oxide (NO) (J Pharmacol Exp Ther 366:265–273, 2018), but little is known about regulation of coronary vasomotor tone by apelin under pathological conditions. Here, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries exposed to cigarette smoke extract (CSE), an established model of second hand smoke exposure. In cultured rat coronary endothelial cells exposed to CSE for 4 hrs, immunoblot analysis demonstrated increased expression of GRK2, which is known to inhibit eNOS activity and NO production in endothelial cells. Rat isolated coronary arterial rings were suspended in myographs in the absence (vehicle control) and presence of CSE for 4 hrs. In rings contracted with 5‐HT (10 ⁻⁷ M), apelin (10 ⁻⁹ ‐10 ⁻⁶ M) caused relaxation of control coronary arteries (pD ₂ =7.41 ± 0.28; E _max = 50 ± 6% relaxation), but had no effect in rings exposed to CSE. By contrast, treatment with CSE had no effect on endothelium‐dependent relaxations to CMF‐019 (10 ⁻⁹ ‐10 ⁻⁵ M), an APJ receptor biased agonist that acts selectively through the G‐protein‐dependent pathway with little effect on GRK2. Endothelium‐dependent relaxation to acetylcholine (ACh) (10 ⁻⁹ ‐10 ⁻⁶ M) was similar in control and CSE‐treated arteries; however, in the presence of apelin (10 ⁻⁷ M), the response to ACh was markedly impaired in arteries exposed to CSE (pD ₂ =7.13 ± 0.22 vs 6.57 ± 0.05, and E _max = 93 ± 4 vs 76 ± 5% relaxation, in the absence and presence of apelin, respectively; n=5; p−7 M) in either control or CSE‐treated coronary arteries. Moreover, apelin (10 ⁻⁷ M) had no effect on relaxation to the NO‐donor, DEA NONOate (10 ⁻⁹ ‐10 ⁻⁴ M), in either control or CSE‐treated arteries. Taken together, the data indicate that apelin failed to cause relaxation in coronary arteries exposed to CSE, likely due to impaired production or release of NO from endothelial cells rather than interfering with the action of NO on coronary smooth muscle cells. Following exposure of coronary arteries to CSE, apelin instead actively inhibited relaxation to another endothelium‐dependent vasodilator; i.e. ACh. That the effects of the G‐protein biased agonist, CMF‐019, did not differ in control and CSE‐treated coronary arteries is consistent with a role for GRK2 activation in the altered response to apelin in CSE‐treated arteries. APJ receptor signaling via the GRK2 pathway may contribute to both the loss of relaxation to apelin itself as well as the ability of apelin to inhibit endothelium‐dependent relaxation to ACh in CSE‐treated coronary arteries. Support or Funding Information: Supported by NHLBI (HL124338)