Special Issue "Sympathetic Nerves and Cardiovascular Diseases".

Cardiovascular diseases (CVDs) constitute a spectrum of disorders affecting the heart and blood vessels, which include coronary heart disease, cerebrovascular disease, and peripheral artery disease [...].

Sympathetic Nervous System and Atherosclerosis.

Atherosclerosis is characterized by the narrowing of the arterial lumen due to subendothelial lipid accumulation, with hypercholesterolemia being a major risk factor. Despite the recent advances in effective lipid-lowering therapies, atherosclerosis remains the leading cause of mortality globally, highlighting the need for additional therapeutic strategies. Accumulating evidence suggests that the sympathetic nervous system plays an important role in atherosclerosis. In this article, we reviewed the sympathetic innervation in the vasculature, norepinephrine synthesis and metabolism, sympathetic activity measurement, and common signaling pathways of sympathetic activation. The focus of this paper was to review the effectiveness of pharmacological antagonists or agonists of adrenoceptors (α1, α2, ß1, ß2, and ß3) and renal denervation on atherosclerosis. All five types of adrenoceptors are present in arterial blood vessels. α1 blockers inhibit atherosclerosis but increase the risk of heart failure while α2 agonism may protect against atherosclerosis and newer generations of ß blockers and ß3 agonists are promising therapies against atherosclerosis; however, new randomized controlled trials are warranted to investigate the effectiveness of these therapies in atherosclerosis inhibition and cardiovascular risk reduction in the future. The role of renal denervation in atherosclerosis inhibition in humans is yet to be established.

Novel AT2R agonist, ß-Pro7Ang III, is cardio- and vaso-protective in diabetic spontaneously hypertensive rats.

Stimulation of the angiotensin II type 2 receptor (AT2R) evokes protective effects in various cardiovascular diseases. Thus, this study aimed to investigate the effects of AT2R stimulation, with or without AT1R blockade, in a model of hypertension with concomitant type 1 diabetes mellitus (T1DM). Spontaneously hypertensive rats (SHRs) were given either citrate or a single dose of streptozotocin (STZ; 55 mg/kg, i.p.) to induce diabetes. After 4 weeks of diabetes, animals were administered either a vehicle (saline), AT2R agonist, ß-Pro7Ang III (0.1 mg/kg/day via osmotic mini-pump), AT1R blocker, candesartan (2 mg/kg/day via drinking water), or a combination of both for a further 8 weeks. ß-Pro7Ang III treatment had no effect on blood pressure, but attenuated the significant increase in cardiac interstitial collagen and protein expression of fibrotic and inflammatory markers, and superoxide levels that was evident in diabetic SHRs. These effects were not observed with candesartan, despite its blood pressure lowering effects. Although ß-Pro7Ang III had no effect on aortic fibrosis, it significantly attenuated MCP-1 protein expression and superoxide levels when compared to both the non-diabetic and diabetic SHRs, to a similar extent as candesartan. In both the heart and vasculature, the effects of ß-Pro7Ang III in combination with candesartan were similar to those of ß-Pro7Ang III alone, and superior to candesartan alone. It was concluded that in hypertension with concomitant diabetes, AT2R stimulation with a novel ligand alone, or in combination with AT1R blockade, improved the cardiac and vascular structural changes that were strongly associated with inflammation and oxidative stress, independent of blood pressure regulation.

Brief early life angiotensin-converting enzyme inhibition attenuates the diuretic response to saline loading in sheep with solitary functioning kidney.

A solitary functioning kidney (SFK) from birth predisposes to hypertension and kidney dysfunction, and this may be associated with impaired fluid and sodium homeostasis. Brief and early angiotensin-converting enzyme inhibition (ACEi) in a sheep model of SFK delays onset of kidney dysfunction. We hypothesized that modulation of the renin-angiotensin system via brief postnatal ACEi in SFK would reprogram renal sodium and water handling. Here, blood pressure (BP), kidney haemodynamics and kidney excretory function were examined in response to an isotonic saline load (0.13 ml/kg/min, 180 min) at 20 months of age in SFK (fetal unilateral nephrectomy at 100 days gestation; term 150 days), sham and SFK+ACEi sheep (ACEi in SFK 4-8 weeks of age). Basal BP was higher in SFK than sham (∼13 mmHg), and similar between SFK and SFK+ACEi groups. Saline loading caused a small increase in BP (∼3-4 mmHg) the first 2 h in SFK and sham sheep but not SFK+ACEi sheep. Glomerular filtration rate did not change in response to saline loading. Total sodium excretion was similar between groups. Total urine excretion was similar between SFK and sham animals but was ∼40% less in SFK+ACEi animals compared with SFK animals. In conclusion, the present study indicates that water homeostasis in response to a physiological challenge is attenuated at 20 months of age by brief early life ACEi in SFK. Further studies are required to determine if ACEi in early life in children with SFK could compromise fluid homeostasis later in life.

Sex- and age-related differences in renal and cardiac injury and senescence in stroke-prone spontaneously hypertensive rats.

BACKGROUND: Sex differences play a critical role in the incidence and severity of cardiovascular diseases, whereby men are at a higher risk of developing cardiovascular disease compared to age-matched premenopausal women. Marked sex differences at the cellular and tissue level may contribute to susceptibility to cardiovascular disease and end-organ damage. In this study, we have performed an in-depth histological analysis of sex differences in hypertensive cardiac and renal injury in middle-aged stroke-prone spontaneously hypertensive rats (SHRSPs) to determine the interaction between age, sex and cell senescence. METHODS: Kidneys, hearts and urine samples were collected from 6.5- and 8-month-old (Mo) male and female SHRSPs. Urine samples were assayed for albumin and creatinine content. Kidneys and hearts were screened for a suite of cellular senescence markers (senescence-associated ß-galactosidase, p16INK4a, p21, γH2AX). Renal and cardiac fibrosis was quantified using Masson's trichrome staining, and glomerular hypertrophy and sclerosis were quantified using Periodic acid-Schiff staining. RESULTS: Marked renal and cardiac fibrosis, concomitant with albuminuria, were evident in all SHRSPs. These sequelae were differentially affected by age, sex and organ. That is, the level of fibrosis was greater in the kidney than the heart, males had greater levels of fibrosis than females in both the heart and kidney, and even a 6-week increase in age resulted in greater levels of kidney fibrosis in males. The differences in kidney fibrosis were reflected by elevated levels of cellular senescence in the kidney in males but not females. Senescent cell burden was significantly less in cardiac tissue compared to renal tissue and was not affected by age or sex. CONCLUSIONS: Our study demonstrates a clear sex pattern in age-related progression of renal and cardiac fibrosis and cellular senescence in SHRSP rats. A 6-week time frame was associated with increased indices of cardiac and renal fibrosis and cellular senescence in male SHRSPs. Female SHRSP rats were protected from renal and cardiac damage compared to age-matched males. Thus, the SHRSP is an ideal model to investigate the effects of sex and aging on organ injury over a short timeframe.

Kidney and cardiovascular diseases are some of the leading causes of death worldwide, and they affect men and women differently. Young men are generally at higher risk of developing these diseases than young women. Women also have unique risk factors for kidney and cardiovascular disease. These may include complications associated with pregnancy, such as preeclampsia, and menopause. For example, the risk of disease for women increases significantly after menopause. In addition, treatment strategies for kidney and cardiovascular diseases are often less effective in women compared to men, but the causes for this are unknown. More research is needed to understand sex differences in kidney and cardiovascular diseases, so that we can develop new drugs that are effective in women as well as men. In this study, we have examined kidney and heart damage associated with elevated blood pressure in adult male rats and adult female rats (long before the onset of menopause). We have shown that males develop significantly more scarring of their hearts and kidneys compared to females. We also identified the cells in the kidneys of male rats, but not female rats, showed signs of DNA damage and early ageing. This suggests cellular damage in young males may contribute to their more rapid progression of kidney disease compared to females. Future research examining females after menopause, when disease risk is greater, will enhance our understanding of cell damage in kidney and cardiovascular disease.

Antihypertensives and Antibiotics: Impact on Intestinal Dysfunction and Hypertension.

Gut dysfunction has emerged as a contributor to hypertension, the leading risk factor for disease globally, including stroke, heart failure, and kidney disease. This is underpinned by breakdown of the homeostatic relationship connecting intestinal epithelial function, the microbiota and immune responses. Antihypertensive medications have been shown to reverse intestinal dysfunction and gut dysbiosis. However, the mechanisms underlying this restoration of gut structure and function remain largely unknown. In this review, we examine current knowledge supporting a role for impaired intestinal epithelial permeability in hypertension, focusing on electrolyte movement, the renin-angiotensin-aldosterone system, and the restorative effects of orally administered antihypertensive medications and antibiotics. Further work is required to determine if the association between intestinal dysfunction and hypertension is causal. This is a rapidly evolving field, with intestinal dysfunction and dysbiosis representing an area that may be exploited to improve treatment of hypertension and cardiovascular disease.

Beneficial effects of brief early life angiotensin-converting enzyme inhibition wane with time in sheep with solitary functioning kidney.

A child with a congenital solitary functioning kidney (SFK) may develop kidney disease from early in life due to hyperfiltration injury. Previously, we showed in a sheep model of SFK that brief angiotensin-converting enzyme inhibition (ACEi) early in life is reno-protective and increases renal functional reserve (RFR) at 8 months of age. Here we investigated the long-term effects of brief early ACEi in SFK sheep out to 20 months of age. At 100 days gestation (term = 150 days) SFK was induced by fetal unilateral nephrectomy, or sham surgery was performed (controls). SFK lambs received enalapril (SFK+ACEi; 0.5 mg/kg, once daily, orally) or vehicle (SFK) from 4 to 8 weeks of age. At 8, 14 and 20 months of age urinary albumin excretion was measured. At 20 months of age, we examined basal kidney function and RFR via infusion of combined amino acid and dopamine (AA+D). SFK+ACEi resulted in lower albuminuria (∼40%) at 8 months, but not at 14 or 20 months of age compared with vehicle-SFK. At 20 months, basal GFR (∼13%) was lower in SFK+ACEi compared with SFK, but renal blood flow (RBF), renal vascular resistance (RVR) and filtration fraction were similar to SFK. During AA+D, the increase in GFR was similar in SFK+ACEi and SFK animals, but the increase in RBF was greater (∼46%) in SFK+ACEi than SFK animals. Brief ACEi in SFK delayed kidney disease in the short-term but these effects were not sustained long-term.

Moxonidine Increases Uptake of Oxidised Low-Density Lipoprotein in Cultured Vascular Smooth Muscle Cells and Inhibits Atherosclerosis in Apolipoprotein E-Deficient Mice.

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE-/-) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE-/- mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE-/- mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

The Novel AT2 Receptor Agonist ß-Pro7-AngIII Exerts Cardiac and Renal Anti-Fibrotic and Anti-Inflammatory Effects in High Salt-Fed Mice.

A high salt (HS) diet is associated with an increased risk for cardiovascular diseases (CVDs) and fibrosis is a key contributor to the organ dysfunction involved in CVDs. The activation of the renin angiotensin type 2 receptor (AT2R) has been considered as organ protective in many CVDs. However, there are limited AT2R-selective agonists available. Our first reported ß-substituted angiotensin III peptide, ß-Pro7-AngIII, showed high selectivity for the AT2R. In the current study, we examine the potential anti-fibrotic and anti-inflammatory effects of this novel AT2R-selective peptide on HS-induced organ damage. FVB/N mice fed with a 5% HS diet for 8 weeks developed cardiac and renal fibrosis and inflammation, which were associated with increased TGF-ß1 levels in heart, kidney and plasma. Four weeks' treatment (from weeks 5-8) with ß-Pro7-AngIII inhibited the HS-induced cardiac and renal fibrosis and inflammation. These protective effects were accompanied by reduced local and systemic TGF-ß1 as well as reduced cardiac myofibroblast differentiation. Importantly, the anti-fibrotic and anti-inflammatory effects caused by ß-Pro7-AngIII were attenuated by the AT2R antagonist PD123319. These results demonstrate, for the first time, the cardio- and reno-protective roles of the AT2R-selective ß-Pro7-AngIII, highlighting it as an important therapeutic that can target the AT2R to treat end-organ damage.

Bezlotoxumab prevents extraintestinal organ damage induced by Clostridioides difficile infection.

Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4+CD8+ thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic.Abbreviations: Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).

Strategies for immortalisation of amnion-derived mesenchymal and epithelial cells.

Mesenchymal (human amniotic mesenchymal stem cell [HAMSC]) and epithelial cells (human amnion epithelial cell [HAEC]) derived from human amniotic membranes possess characteristics of pluripotency. However, the pluripotency of HAMSC and HAEC are sustained only for a finite period. This in vitro cell growth can be extended by cell immortalisation. Many well-defined immortalisation systems have been used for artificially overexpressing genes such as human telomerase reverse transcriptase in HAMSC and HAEC leading to controlled and prolonged cell proliferation. In recent years, much progress has been made in our understanding of the cellular machinery that regulates the cell cycle when immortalised. This review summarises the current understanding of molecular mechanisms that contribute to cell immortalisation, the strategies that have been employed to immortalise amnion-derived cell types, and their likely applications in regenerative medicine.

Kidney Angiotensin in Cardiovascular Disease: Formation and Drug Targeting.

The concept of local formation of angiotensin II in the kidney has changed over the last 10-15 years. Local synthesis of angiotensinogen in the proximal tubule has been proposed, combined with prorenin synthesis in the collecting duct. Binding of prorenin via the so-called (pro)renin receptor has been introduced, as well as megalin-mediated uptake of filtered plasma-derived renin-angiotensin system (RAS) components. Moreover, angiotensin metabolites other than angiotensin II [notably angiotensin-(1-7)] exist, and angiotensins exert their effects via three different receptors, of which angiotensin II type 2 and Mas receptors are considered renoprotective, possibly in a sex-specific manner, whereas angiotensin II type 1 (AT1) receptors are believed to be deleterious. Additionally, internalized angiotensin II may stimulate intracellular receptors. Angiotensin-converting enzyme 2 (ACE2) not only generates angiotensin-(1-7) but also acts as coronavirus receptor. Multiple, if not all, cardiovascular diseases involve the kidney RAS, with renal AT1 receptors often being claimed to exert a crucial role. Urinary RAS component levels, depending on filtration, reabsorption, and local release, are believed to reflect renal RAS activity. Finally, both existing drugs (RAS inhibitors, cyclooxygenase inhibitors) and novel drugs (angiotensin receptor/neprilysin inhibitors, sodium-glucose cotransporter-2 inhibitors, soluble ACE2) affect renal angiotensin formation, thereby displaying cardiovascular efficacy. Particular in the case of the latter three, an important question is to what degree they induce renoprotection (e.g., in a renal RAS-dependent manner). This review provides a unifying view, explaining not only how kidney angiotensin formation occurs and how it is affected by drugs but also why drugs are renoprotective when altering the renal RAS. SIGNIFICANCE STATEMENT: Angiotensin formation in the kidney is widely accepted but little understood, and multiple, often contrasting concepts have been put forward over the last two decades. This paper offers a unifying view, simultaneously explaining how existing and novel drugs exert renoprotection by interfering with kidney angiotensin formation.

Brief Early Life Angiotensin-Converting Enzyme Inhibition Offers Renoprotection in Sheep with a Solitary Functioning Kidney at 8 Months of Age.

BACKGROUND: Children born with a solitary functioning kidney (SFK) are predisposed to develop hypertension and kidney injury. Glomerular hyperfiltration and hypertrophy contribute to the pathophysiology of kidney injury. Angiotensin-converting enzyme inhibition (ACEi) can mitigate hyperfiltration and may be therapeutically beneficial in reducing progression of kidney injury in those with an SFK. METHODS: SFK was induced in male sheep fetuses at 100 days gestation (term=150 days). Between 4 and 8 weeks of age, SFK lambs received enalapril (SFK+ACEi; 0.5mg/kg per day, once daily, orally) or vehicle (SFK). At 8 months, we examined BP, basal kidney function, renal functional reserve (RFR; GFR response to combined amino acid and dopamine infusion), GFR response to nitric oxide synthase (NOS) inhibition, and basal nitric oxide (NO) bioavailability (basal urinary total nitrate and nitrite [NOx]). RESULTS: SFK+ACEi prevented albuminuria and resulted in lower basal GFR (16%), higher renal blood flow (approximately 22%), and lower filtration fraction (approximately 35%), but similar BP, compared with vehicle-treated SFK sheep. Together with greater recruitment of RFR (approximately 14%) in SFK+ACEi than SFK animals, this indicates a reduction in glomerular hyperfiltration-mediated kidney dysfunction. During NOS inhibition, the decrease in GFR (approximately 14%) was greater among SFK+ACEi than among SFK animals. Increased (approximately 85%) basal urinary total NOx in SFK+ACEi compared with SFK animals indicates elevated NO bioavailability likely contributed to improvements in kidney function and prevention of albuminuria. CONCLUSIONS: Brief and early ACEi in SFK is associated with reduced glomerular hyperfiltration-mediated kidney disease up to 8 months of age in a sheep model.

Renal Denervation in Combination With Angiotensin Receptor Blockade Prolongs Blood Pressure Trough During Hemorrhage.

Majority of patients with hypertension and chronic kidney disease (CKD) undergoing renal denervation (RDN) are maintained on antihypertensive medication. However, RDN may impair compensatory responses to hypotension induced by blood loss. Therefore, continuation of antihypertensive medications in denervated patients may exacerbate hypotensive episodes. This study examined whether antihypertensive medication compromised hemodynamic responses to blood loss in normotensive (control) sheep and in sheep with hypertensive CKD at 30 months after RDN (control-RDN, CKD-RDN) or sham (control-intact, CKD-intact) procedure. CKD-RDN sheep had lower basal blood pressure (BP; ≈9 mm Hg) and higher basal renal blood flow (≈38%) than CKD-intact. Candesartan lowered BP and increased renal blood flow in all groups. 10% loss of blood volume alone caused a modest fall in BP (≈6-8 mm Hg) in all groups but did not affect the recovery of BP. 10% loss of blood volume in the presence of candesartan prolonged the time at trough BP by 9 minutes and attenuated the fall in renal blood flow in the CKD-RDN group compared with CKD-intact. Candesartan in combination with RDN prolonged trough BP and attenuated renal hemodynamic responses to blood loss. To minimize the risk of hypotension-mediated organ damage, patients with RDN maintained on antihypertensive medications may require closer monitoring when undergoing surgery or experiencing traumatic blood loss.

Adjustment for body mass index changes inverse associations of HDL-cholesterol with blood pressure and hypertension to positive associations.

The associations between high-density lipoprotein cholesterol (HDL-C) and blood pressure (BP) or hypertension are inconsistent in previous studies. This study aimed to assess these associations in a large cohort of Chinese adults and across different age groups. This cross-sectional association study included 22,081 Chinese adults. Associations of HDL-C with BP and hypertension were analyzed using linear or logistic regression, with or without adjustment for confounding factors. HDL-C was inversely associated with BP and hypertension. These associations were still apparent after adjustment for age, sex, fasting plasma glucose, and low-density lipoprotein cholesterol. Sub-analyses revealed: (1) in the whole cohort and females alone, HDL-C was inversely associated with BP and hypertension in young and middle-aged but not older participants; (2) in males alone, HDL-C was not associated with systolic BP or hypertension. However, HDL-C was either inversely, or not, or positively associated with BP in young, middle-aged, and older males, respectively. After further adjustment for body mass index (BMI), the negative associations of HDL-C with BP and hypertension in the whole cohort became positive ones, and the positive associations only presented in males. These findings suggest that further adjustment for BMI changes inverse associations of HDL-cholesterol with BP and hypertension to positive associations in a cohort of Chinese adults.

Renal Deafferentation Prevents Progression of Hypertension and Changes to Sympathetic Reflexes in a Rabbit Model of Chronic Kidney Disease.

[Figure: see text].

Blunted natriuretic response to saline loading in sheep with hypertensive kidney disease following radiofrequency catheter-based renal denervation.

Renal sympathetic nerves contribute to renal excretory function during volume expansion. We hypothesized that intact renal innervation is required for excretion of a fluid/electrolyte load in hypertensive chronic kidney disease (CKD) and normotensive healthy settings. Blood pressure, kidney hemodynamic and excretory response to 180 min of isotonic saline loading (0.13 ml/kg/min) were examined in female normotensive (control) and hypertensive CKD sheep at 2 and 11 months after sham (control-intact, CKD-intact) or radiofrequency catheter-based RDN (control-RDN, CKD-RDN) procedure. Basal blood pressure was ~ 7 to 9 mmHg lower at 2, and 11 months in CKD-RDN compared with CKD-intact sheep. Saline loading did not alter glomerular filtration rate in any group. At 2 months, in response to saline loading, total urine and sodium excretion were ~ 40 to 50% less, in control-RDN and CKD-RDN than intact groups. At 11 months, the natriuretic and diuretic response to saline loading were similar between control-intact, control-RDN and CKD-intact groups but sodium excretion was ~ 42% less in CKD-RDN compared with CKD-intact at this time-point. These findings indicate that chronic withdrawal of basal renal sympathetic activity impairs fluid/electrolyte excretion during volume expansion. Clinically, a reduced ability to excrete a saline load following RDN may contribute to disturbances in body fluid balance in hypertensive CKD.