Cardiovascular dysfunction induced by combined exposure to nicotine inhalation and high-fat diet.

Smoking and high-fat diet (HFD) consumption are two modifiable risk factors for cardiovascular (CV) diseases, and individuals who are overweight or obese due to unhealthy diet are more likely to use tobacco products. In this study, we aim to investigate the combined effects of nicotine (the addictive component of all tobacco products) and HFD on CV health, which are poorly understood. C57BL/6N male mice were placed on either HFD (60 kcal% fat) or regular diet (22 kcal% fat) and exposed to air or nicotine vapor for 10-12 wk. CV function was monitored by echocardiography and radiotelemetry, with left ventricular (LV) catheterization and aortic ring vasoreactivity assays performed at end point. Mice on HFD exhibited increased heart rate and impaired parasympathetic tone, whereas nicotine exposure increased sympathetic vascular tone as evidenced by increased blood pressure (BP) response to ganglionic blockade. Although neither nicotine nor HFD alone or in combination significantly altered BP, nicotine exposure disrupted circadian BP regulation with reduced BP dipping. LV catheterization revealed that combined exposure to nicotine and HFD led to LV diastolic dysfunction with increased LV end-diastolic pressure (LVEDP). Moreover, combined exposure resulted in increased inhibitory phosphorylation of endothelial nitric oxide synthase and greater impairment of endothelium-dependent vasodilation. Finally, a small cohort of C57BL/6N females with combined exposure exhibited similar increases in LVEDP, indicating that both sexes are susceptible to the combined effect of nicotine and HFD. In summary, combined exposure to nicotine and HFD leads to greater CV harm, including both additive and new-onset CV dysfunction.NEW & NOTEWORTHY Nicotine product usage and high-fat diet consumption are two modifiable risk factors for cardiovascular diseases. Here, we demonstrate that in mice, combined exposure to inhaled nicotine and high-fat diet results in unique cardiovascular consequences compared with either treatment alone, including left ventricular diastolic dysfunction, dysregulation of blood pressure, autonomic dysfunction, and greater impairment of endothelium-dependent vasorelaxation. These findings indicate that individuals who consume both nicotine products and high-fat diet have distinctive cardiovascular risks.

Ovarian hormones do not mediate protection against pulmonary hypertension and right ventricular remodeling in female mice exposed to chronic, inhaled nicotine.

Electronic cigarette use has increased globally prompting calls for improved understanding of nicotine's cardiovascular health effects. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular (RV) remodeling in male mice, but not female mice, suggesting sex differences in nicotine-related pathology. Clinically, biological females develop pulmonary hypertension more often but have less severe disease than biological males, likely because of the cardiopulmonary protective effects of estrogen. Nicotine is also metabolized more rapidly in biological females because of differences in cytochrome-P450 activity, which are thought to be mediated by female sex hormones. These findings led us to hypothesize that female mice are protected against nicotine-induced pulmonary hypertension by an ovarian hormone-dependent mechanism. In this study, intact and ovariectomized (OVX) female mice were exposed to chronic, inhaled nicotine or room air for 12 h/day for 10-12 wk. We report no differences in serum cotinine levels between intact and OVX mice. In addition, we found no structural (RV or left ventricular dimensions and Fulton index) or functional (RV systolic pressure, pulmonary vascular resistance, cardiac output, ejection fraction, and fractional shortening) evidence of cardiopulmonary dysfunction in intact or OVX mice. We conclude that ovarian hormones do not mediate cardiopulmonary protection against nicotine-induced pulmonary hypertension. Due to profound sex differences in clinical pulmonary hypertension pathogenesis and nicotine metabolism, further studies are necessary to elucidate mechanisms underlying protection from nicotine-induced pathology in female mice.NEW & NOTEWORTHY The emergence of electronic cigarettes poses a threat to cardiovascular and pulmonary health, but the direct contribution of nicotine to these disease processes is largely unknown. Our laboratory has previously shown that chronic, inhaled nicotine induces pulmonary hypertension and right ventricular remodeling in male mice, but not female mice. This study using a bilateral ovariectomy model suggests that the cardiopulmonary protection observed in nicotine-exposed female mice may be independent of ovarian hormones.

Alpha7 nicotinic acetylcholine receptor mediates chronic nicotine inhalation-induced cardiopulmonary dysfunction.

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.

Sex-Dependent Effects of Inhaled Nicotine on the Gut Microbiome.

INTRODUCTION: The impact of nicotine, the addictive component of both traditional cigarettes and e-cigarettes, on many physiological processes remains poorly understood. To date, there have been few investigations into the impact of nicotine on the gut microbiome, and these studies utilized oral administration rather than inhalation. This study aimed to establish if inhaled nicotine alters the gut microbiome and the effect of sex as a biological variable. METHODS: Female (n = 8 air; n = 10 nicotine) and male (n = 10 air; n = 10 nicotine) C57BL6/J mice were exposed to air (control) or nicotine vapor (12 hour/day) for 13 weeks. A fecal sample was collected from each mouse at the time of sacrifice, and the gut microbiome was analyzed by 16S rRNA gene sequencing. QIIME2, PICRUSt, and STAMP were used to detect gut bacterial differences and functional metabolic pathways. RESULTS: Sex-specific differences were observed in both alpha and beta diversities in the absence of nicotine. While nicotine alters microbial community structure in both male and female mice as revealed by the beta diversity metric, nicotine significantly reduced alpha diversity only in female mice. A total of 42 bacterial taxa from phylum to species were found to be significantly different among the treatment groups. Finally, analysis for functional genes revealed significant differences in twelve metabolic pathways in female mice and ten in male mice exposed to nicotine compared to air controls. CONCLUSIONS: Nicotine inhalation alters the gut microbiome and reduces bacterial diversity in a sex-specific manner, which may contribute to the overall adverse health impact of nicotine. IMPLICATIONS: The gut microbiota plays a fundamental role in the well-being of the host, and traditional cigarette smoking has been shown to affect the gut microbiome. The effects of nicotine alone, however, remain largely uncharacterized. Our study demonstrates that nicotine inhalation alters the gut microbiome in a sex-specific manner, which may contribute to the adverse health consequences of inhaled nicotine. This study points to the importance of more detailed investigations into the influence of inhaled nicotine on the gut microbiota.

Angiotensin II type 1 receptor mediates pulmonary hypertension and right ventricular remodeling induced by inhaled nicotine.

Use of electronic cigarettes is rapidly increasing among youth and young adults, but little is known regarding the long-term cardiopulmonary health impacts of these nicotine-containing devices. Our group has previously demonstrated that chronic, inhaled nicotine induces pulmonary hypertension (PH) and right ventricular (RV) remodeling in mice. These changes were associated with upregulated RV angiotensin-converting enzyme (ACE). Angiotensin II receptor blockers (ARBs) have been shown to reverse cigarette smoking-induced PH in rats. ACE inhibitor and ARB use in a large retrospective cohort of patients with PH is associated with improved survival. Here, we utilized losartan (an ARB specific for angiotensin II type 1 receptor) to further explore nicotine-induced PH. Male C57BL/6 mice received nicotine vapor for 12 h/day, and exposure was assessed using serum cotinine to achieve levels comparable to human smokers or electronic cigarette users. Mice were exposed to nicotine for 8 wk and a subset was treated with losartan via an osmotic minipump. Cardiac function was assessed using echocardiography and catheterization. Although nicotine exposure increased angiotensin II in the RV and lung, this finding was nonsignificant. Chronic, inhaled nicotine significantly increased RV systolic pressure and RV free wall thickness versus air control. These parameters were significantly lower in mice receiving both nicotine and losartan. Nicotine significantly increased RV internal diameter, with no differences seen between the nicotine and nicotine-losartan group. Neither nicotine nor losartan affected left ventricular structure or function. These findings provide the first evidence that antagonism of the angiotensin II type 1 receptor can ameliorate chronic, inhaled nicotine-induced PH and RV remodeling.NEW & NOTEWORTHY Chronic, inhaled nicotine causes pulmonary hypertension and right ventricular remodeling in mice. Treatment with losartan, an angiotensin II type 1 receptor antagonist, ameliorates nicotine-induced pulmonary hypertension and right ventricular remodeling. This novel finding provides preclinical evidence for the use of renin-angiotensin system-based therapies in the treatment of pulmonary hypertension, particularly in patients with a history of tobacco-product use.

Loss of endothelial sulfatase-1 after experimental sepsis attenuates subsequent pulmonary inflammatory responses.

Sepsis patients are at increased risk for hospital-acquired pulmonary infections, potentially due to postseptic immunosuppression known as the compensatory anti-inflammatory response syndrome (CARS). CARS has been attributed to leukocyte dysfunction, with an unclear role for endothelial cells. The pulmonary circulation is lined by an endothelial glycocalyx, a heparan sulfate-rich layer essential to pulmonary homeostasis. Heparan sulfate degradation occurs early in sepsis, leading to lung injury. Endothelial synthesis of new heparan sulfates subsequently allows for glycocalyx reconstitution and endothelial recovery. We hypothesized that remodeling of the reconstituted endothelial glycocalyx, mediated by alterations in the endothelial machinery responsible for heparan sulfate synthesis, contributes to CARS. Seventy-two hours after experimental sepsis, coincident with glycocalyx reconstitution, mice demonstrated impaired neutrophil and protein influx in response to intratracheal lipopolysaccharide (LPS). The postseptic reconstituted glycocalyx was structurally remodeled, with enrichment of heparan sulfate disaccharides sulfated at the 6-O position of glucosamine. Increased 6-O-sulfation coincided with loss of endothelial sulfatase-1 (Sulf-1), an enzyme that specifically removes 6-O-sulfates from heparan sulfate. Intravenous administration of Sulf-1 to postseptic mice restored the pulmonary response to LPS, suggesting that loss of Sulf-1 was necessary for postseptic suppression of pulmonary inflammation. Endothelial-specific knockout mice demonstrated that loss of Sulf-1 was not sufficient to induce immunosuppression in non-septic mice. Knockdown of Sulf-1 in human pulmonary microvascular endothelial cells resulted in downregulation of the adhesion molecule ICAM-1. Taken together, our study indicates that loss of endothelial Sulf-1 is necessary for postseptic suppression of pulmonary inflammation, representing a novel endothelial contributor to CARS.

Differential Protein Expression Profiles of Bronchoalveolar Lavage Fluid Following Lipopolysaccharide-Induced Direct and Indirect Lung Injury in Mice.

The pathogenic mechanisms of acute lung injury due to direct and indirect pulmonary insults are incompletely understood. Using an unbiased, discovery and quantitative proteomic approach, we examined bronchoalveolar lavage fluid (BALF) proteome following lipopolysaccharide (LPS)-induced direct and indirect lung injury in mice. A total of 1017 proteins were both identified and quantitated in BALF from control, intratracheal (I.T., direct) and intraperitoneal (I.P., indirect) LPS-treated mice. The two LPS groups shared 13 up-regulated and 22 down-regulated proteins compared to the control group. Ingenuity pathway analysis revealed that acute-phase response signaling was activated by both I.T. and I.P. LPS; however, the magnitude of activation was much greater in the I.T. LPS group. Intriguingly, two canonical signaling pathways, liver X receptor/retinoid X receptor activation, and the production of nitric oxide and reactive oxygen species in macrophages, were activated by I.T. but suppressed by I.P. LPS. Cxcl15 (also known as lungkine) was also up-regulated by I.T. but down-regulated by I.P. LPS. In conclusion, our quantitative discovery-based proteomic approach identified commonalities, as well as significant differences in BALF protein expression profiles between LPS-induced direct and indirect lung injury, and importantly, LPS-induced indirect lung injury resulted in suppression of select components of lung innate immunity.

Nicotine and the renin-angiotensin system.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases (CVPD). Although cigarette smoking has been in constant decline since the 1950s, the introduction of e-cigarettes or electronic nicotine delivery systems 10 yr ago has attracted former smokers as well as a new generation of consumers. Nicotine is a highly addictive substance, and it is currently unclear whether e-cigarettes are "safer" than regular cigarettes or whether they have the potential to reverse the health benefits, notably on the cardiopulmonary system, acquired with the decline of tobacco smoking. Of great concern, nicotine inhalation devices are becoming popular among young adults and youths, emphasizing the need for awareness and further study of the potential cardiopulmonary risks of nicotine and associated products. This review focuses on the interaction between nicotine and the renin-angiotensin system (RAS), one of the most important regulatory systems on autonomic, cardiovascular, and pulmonary functions in both health and disease. The literature presented in this review strongly suggests that nicotine alters the homeostasis of the RAS by upregulating the detrimental angiotensin-converting enzyme (ACE)/angiotensin (ANG)-II/ANG II type 1 receptor axis and downregulating the compensatory ACE2/ANG-(1-7)/Mas receptor axis, contributing to the development of CVPD.

Epithelial Deletion of Sulf2 Exacerbates Bleomycin-Induced Lung Injury, Inflammation, and Mortality.

Epithelial injury has been proposed to be the initiating factor in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have shown previously that heparan sulfate 6-O-endosulfatase (Sulf) 2 is overexpressed in the hyperplastic type II alveolar epithelial cells (AECs) in the IPF lungs. By removing 6-O-sulfates from specific heparan sulfate intrachain sites, Sulf2 modulates the functions of many growth factors and cytokines. In this study, we hypothesized that Sulf2 plays a regulatory role in alveolar epithelial injury and repair, using the murine bleomycin model. Consistent with our findings in human IPF lungs, bleomycin treatment in mice resulted in up-regulation of Sulf2 mRNA in whole-lung extracts and overexpression of Sulf2 protein in type II AECs on lung tissue sections. Sulf2 protein was detectable in bronchoalveolar lavage fluid at baseline, and its level was significantly increased after bleomycin exposure. To study the role of Sulf2 in alveolar injury and repair in vivo, we generated a doxycycline-inducible epithelial-specific Sulf2 conditional knockout (Sulf2 CKO) mouse line. After bleomycin exposure, Sulf2 CKO mice exhibited enhanced neutrophil infiltration in the lung, with elevated levels of total protein, lactate dehydrogenase, and cytokines (granulocyte colony-stimulating factor and interferon-γ-inducible protein 10) in bronchoalveolar lavage fluid compared with wild-type littermates. We further showed that both the p53-p21 DNA damage response and the transforming growth factor-ß1 signaling pathway were up-regulated in Sulf2 CKO mice compared with wild-type. Finally, Sulf2 CKO mice suffered increased mortality after bleomycin exposure. In conclusion, Sulf2 expression in type II AECs plays a protective role in epithelial injury, inflammation and mortality.

High-fat diet-induced glucose dysregulation is independent of changes in islet ACE2 in mice.

While restoration of ACE2 activity in the pancreas leads to improvement of glycemia in experimental models of Type 2 diabetes, global deficiency in ACE2 disrupts ß-cell function and impairs glucose tolerance in mice, demonstrating the physiological role of ACE2 in glucose homeostasis. Although the contribution of pancreatic ACE2 to glucose regulation has been demonstrated in genetic models of diabetes and in models with overexpression of the renin-angiotensin system (RAS), it is unclear whether islet ACE2 is involved in glycemic control in common models of human Type 2 diabetes. To determine whether diet-induced diabetes deregulates glucose homeostasis via reduction of ACE2 in the pancreatic islets, wild-type (WT) and ACE2 knockout (KO) male mice were fed a high-fat diet (HFD) for 16 wk. ACE2 KO mice were more susceptible than WT mice to HFD-mediated glycemic dysregulation. Islet ACE2 activity and expression of various genes, including ANG II type 1a receptor (mAT1aR) were then assessed. Surprisingly, we observed no change in islet ACE2 activity and expression despite local RAS overactivity, indicated by an upregulation of mAT1aR expression. Despite a predominant expression in islet α-cells, further investigation highlighted a minor role for ACE2 on glucagon expression. Further, pancreatic ACE2 gene therapy improved glycemia in HFD-fed WT mice, leading to enhanced glucose-stimulated insulin secretion, reduced pancreatic ANG II levels, fibrosis, and ADAM17 activity. Altogether, our study demonstrates that HFD feeding increases RAS activity and mediates glycemic dysregulation likely through loss of ACE2 present outside the islets but independently of changes in islet ACE2.

Up-regulation of heparan sulfate 6-O-sulfation in idiopathic pulmonary fibrosis.

Heparan sulfate proteoglycans (HSPGs) are integral components of the lung. Changes in HSPGs have been documented in idiopathic pulmonary fibrosis (IPF). Many of the biological functions of HSPGs are mediated by heparan sulfate (HS) side chains, and little is understood about these side chains in the pathogenesis of IPF. The aims of this study were to compare HS structure between normal and IPF lungs and to examine how changes in HS regulate the fibrotic process. HS disaccharide analysis revealed that HS 6-O-sulfation was significantly increased in IPF lungs compared with normal lungs, concomitant with overexpression of HS 6-O-sulfotransferases 1 and 2 (HS6ST1/2) mRNA. Immunohistochemistry revealed that HS6ST2 was specifically expressed in bronchial epithelial cells, including those lining the honeycomb cysts in IPF lungs, whereas HS6ST1 had a broad expression pattern. Lung fibroblasts in the fibroblastic foci of IPF lungs expressed HS6ST1, and overexpression of HS6ST1 mRNA was observed in primary lung fibroblasts isolated from IPF lungs compared with those from normal lungs. In vitro, small interference RNA-mediated silencing of HS6ST1 in primary normal lung fibroblasts resulted in reduced Smad2 expression and activation and in reduced expression of collagen I and α-smooth muscle actin after TGF-ß1 stimulation. Similar results were obtained in primary IPF lung fibroblasts. Furthermore, silencing of HS6ST1 in normal and IPF lung fibroblasts resulted in significant down-regulation of TßRIII (betaglycan). In summary, HS 6-O-sulfation is up-regulated in IPF with overexpression of HS6ST1 and HS6ST2, and overexpression of HS6ST1 in lung fibroblasts may regulate their fibrotic responses to TGF-ß1.

UBR1 Promotes Sex-Dependent ACE2 Ubiquitination in Hypertension.

Background: Angiotensin (Ang)-II impairs the function of the antihypertensive enzyme ACE2 by promoting its internalization, ubiquitination and degradation thus contributing to hypertension. However, few ACE2 ubiquitination partners have been identified and their role in hypertension remains unknown. Methods: Proteomics and bioinformatic analysis were used to identify ACE2 ubiquitination partners in the brain, heart, and kidney from Ang-II-infused C57BL6/J mice from both sexes and validated the interaction between UBR1 and ACE2 in cells. Central and peripheral UBR1 knockdown was then performed in male mice to investigate its role in the maintenance of hypertension. Results: Proteomics analysis from hypothalamus identified UBR1 as a potential E3 ligase promoting ACE2 ubiquitination. Enhanced UBR1 expression, associated with ACE2 reduction, was confirmed in various tissues from hypertensive male mice and human samples. Treatment of endothelial and smooth muscle cells with testosterone, but not 17ß-estradiol, confirmed a sex-specific regulation of UBR1. In vivo silencing of UBR1 using chronic administration of small interference RNA resulted in the restoration of ACE2 levels in hypertensive males. A transient decrease in blood pressure following intracerebroventricular, but not systemic, infusion was also observed. Interestingly, UBR1 knockdown increased the brain activation of Nedd4-2, an E3 ligase promoting ACE2 ubiquitination and reduced expression of SGK1, the kinase inactivating Nedd4-2. Conclusions: These data demonstrate that UBR1 is a novel ubiquitin ligase targeting ACE2 in hypertension. UBR1 and Nedd4-2 E3 ligases appear to work synergistically to ubiquitinate ACE2. Targeting of these ubiquitin ligases may represent a novel strategy to restore ACE2 compensatory activity in hypertension.

Overexpression of Sulf2 in idiopathic pulmonary fibrosis.

Previously, we have shown that heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) is a transforming growth factor-ß1 (TGF-ß1)-responsive gene in normal human lung fibroblasts and functions as a negative feedback regulator of TGF-ß1 and that TGF-ß1 induces the expression of Sulf1 as well as that of the closely related Sulf2 in a murine model of pulmonary fibrosis. In this study, we focused on the role of Sulf2 in modulating TGF-ß1 function and the development of pulmonary fibrosis. We found that Sulf2 mRNA was overexpressed in lung samples from human patients with idiopathic pulmonary fibrosis (IPF), and Sulf2 protein was specifically localized to the hyperplastic type II alveolar epithelial cells (AECs). In vitro, TGF-ß1 induced the expression of Sulf2 with accompanied HS 6-O-desulfation in A549 cells, adenocarcinoma cells derived from the type II alveolar epithelium. Using small interference RNA to block Sulf2 expression, we observed a biphasic TGF-ß1 response with early enhanced Smad activation, but eventually reduced TGF-ß1 target gene expression in Sulf2 knockdown A549 cells compared with the control cells. To study the role of Sulf2 in normal type II AECs, we isolated primary type II cells from wild-type and Sulf2 knockout mice. We observed enhanced Smad activation as well as enhanced TGF-ß1 target gene expression in Sulf2 knockout type II AECs compared with wild-type type II AECs. In conclusion, Sulf2 is overexpressed in IPF and may play a role in regulating TGF-ß1 signaling in type II AECs.

Nedd4-2 up-regulation is associated with ACE2 ubiquitination in hypertension.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension. METHODS AND RESULTS: Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST. CONCLUSION: Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.

Nicotine and novel tobacco products drive adverse cardiac remodeling and dysfunction in preclinical studies.

Background: The heart undergoes structural and functional changes in response to injury and hemodynamic stress known as cardiac remodeling. Cardiac remodeling often decompensates causing dysfunction and heart failure (HF). Cardiac remodeling and dysfunction are significantly associated with cigarette smoking. Although cigarette smoking has declined, the roles of nicotine and novel tobacco products (including electronic cigarettes and heat-not-burn tobacco) in cardiac remodeling are unclear. In this perspective, we present evidence demonstrating maladaptive cardiac remodeling in nicotine-exposed mice undergoing hemodynamic stress with angiotensin (Ang)-II infusion and review preclinical literature linking nicotine and novel tobacco products with cardiac remodeling and dysfunction. Methods: Adult, male C57BL/6J mice were exposed to room air or chronic, inhaled nicotine for 8 weeks. A subset of mice was infused with Ang-II via subcutaneous osmotic mini-pumps during the final 4 weeks of exposure. Left ventricular structure and function were assessed with echocardiography. Results: Chronic, inhaled nicotine abrogated Ang-II-induced thickening of the left ventricular posterior wall, leading to reduced relative wall thickness. Ang-II infusion was associated with increased left ventricular mass index in both air- and nicotine-exposed mice. Conclusions: These changes suggest a phenotypic shift from concentric hypertrophy to eccentric hypertrophy in nicotine-exposed, hemodynamically-stressed mice which could drive HF pathogenesis. These findings join a growing body of animal studies demonstrating cardiac remodeling and dysfunction following nicotine and electronic cigarette exposure. Further exploration is necessary; however, clinicians and researchers should not overlook these emerging products as potential risk factors in the pathogenesis of cardiac remodeling and associated diseases including HF.

Nicotine and vascular dysfunction.

Cigarette smoking is the single most important risk factor for the development of cardiovascular diseases (CVDs). However, the role of nicotine, the addictive component of all tobacco products, in the development of CVD is incompletely understood. Although increased public awareness of the harms of cigarette smoking has successfully led to a decline in its prevalence, the use of electronic cigarettes (e-cig) or electronic nicotine delivery system has increased dramatically in recent years because of the perception that these products are safe. This review summarizes our current knowledge of the expression and function of the nicotinic acetylcholine receptors in the cardiovascular system and the impact of nicotine exposure on cardiovascular health, with a focus on nicotine-induced vascular dysfunction. Nicotine alters vasoreactivity through endothelium-dependent and/or endothelium-independent mechanisms, leading to clinical manifestations in both cigarette smokers and e-cig users. In addition, nicotine induces vascular remodelling through its effects on proliferation, migration and matrix production of both vascular endothelial and vascular smooth muscle cells. The purpose of this review is to identify critical knowledge gaps regarding the effects of nicotine on the vasculature and to stimulate continued nicotine research.

Angiotensin-converting enzyme 2 overexpression in the subfornical organ prevents the angiotensin II-mediated pressor and drinking responses and is associated with angiotensin II type 1 receptor downregulation.

We recently reported the presence of angiotensin-converting enzyme (ACE)2 in brain regions controlling cardiovascular function; however, the role of ACE2 in blood pressure regulation remains unclear because of the lack of specific tools to investigate its function. We hypothesized that ACE2 could play a pivotal role in the central regulation of cardiovascular function by regulating other renin-angiotensin system components. To test this hypothesis, we generated an adenovirus expressing the human ACE2 cDNA upstream of an enhanced green fluorescent protein (eGFP) reporter gene (Ad-hACE2-eGFP). In vitro characterization shows that neuronal cells infected with Ad-hACE2-eGFP (10 to 100 multiplicities of infection), but not Ad-eGFP (100 multiplicities of infection), exhibit dose-dependent ACE2 expression and activity. In addition, an active secreted form was detected in the conditioned medium. In vivo, Ad-hACE2-eGFP infection (2x10(6) plaque-forming units intracerebroventricularly) produced time-dependent expression and activity (with a peak at 7 days) in the mouse subfornical organ. More importantly, 7 days after virus infection, the pressor response to angiotensin (Ang) II (200 pmol intracerebroventricularly) was significantly reduced in Ad-hACE2-eGFP-treated mice compared with controls. Furthermore, subfornical organ-targeted ACE2 overexpression dramatically reduced the Ang II-mediated drinking response. Interestingly, ACE2 overexpression was associated with downregulation of the Ang II type 1 receptor expression both in vitro and in vivo. These data suggest that ACE2 overexpression in the subfornical organ impairs Ang II-mediated pressor and drinking responses at least by inhibiting the Ang II type 1 receptor expression. Taken together, our results show that ACE2 plays a pivotal role in the central regulation of blood pressure and volume homeostasis, offering a new target for the treatment of hypertension and other cardiovascular diseases.

ACE2 mouse models: a toolbox for cardiovascular and pulmonary research.

Angiotensin-converting enzyme 2 (ACE2) has been identified as the host entry receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID-19 pandemic. ACE2 is a regulatory enzyme of the renin-angiotensin system and has protective functions in many cardiovascular, pulmonary and metabolic diseases. This review summarizes available murine models with systemic or organ-specific deletion of ACE2, or with overexpression of murine or human ACE2. The purpose of this review is to provide researchers with the genetic tools available for further understanding of ACE2 biology and for the investigation of ACE2 in the pathogenesis and treatment of COVID-19.

Effects of Chronic Nicotine Inhalation on Systemic and Pulmonary Blood Pressure and Right Ventricular Remodeling in Mice.

Cigarette smoking is the single most important risk factor for the development of cardiovascular and pulmonary diseases; however, the role of nicotine in the pathogenesis of these diseases is incompletely understood. The purpose of this study was to examine the effects of chronic nicotine inhalation on the development of cardiovascular and pulmonary disease with a focus on blood pressure and cardiac remodeling. Male C57BL6/J mice were exposed to air (control) or nicotine vapor (daily, 12 hour on/12 hour off) for 8 weeks. Systemic blood pressure was recorded weekly by radio-telemetry, and cardiac remodeling was monitored by echocardiography. At the end of the 8 weeks, mice were subjected to right heart catheterization to measure right ventricular systolic pressure. Nicotine-exposed mice exhibited elevated systemic blood pressure from weeks 1 to 3, which then returned to baseline from weeks 4 to 8, indicating development of tolerance to nicotine. At 8 weeks, significantly increased right ventricular systolic pressure was detected in nicotine-exposed mice compared with the air controls. Echocardiography showed that 8-week nicotine inhalation resulted in right ventricular (RV) hypertrophy with increased RV free wall thickness and a trend of increase in RV internal diameter. In contrast, there were no significant structural or functional changes in the left ventricle following nicotine exposure. Mechanistically, we observed increased expression of angiotensin-converting enzyme and enhanced activation of mitogen-activated protein kinase pathways in the RV but not in the left ventricle. We conclude that chronic nicotine inhalation alters both systemic and pulmonary blood pressure with the latter accompanied by RV remodeling, possibly leading to progressive and persistent pulmonary hypertension.

Hypoxic induction of myocardial vascularization during development.

The development of the heart is closely linked to its temporally and spatially regulated vascularization. Hypoxia has been shown to stimulate myocardial capillary growth and improve myocardial perfusion during reperfusion in postnatal animals exposed to chronic or intermittent exposure to hypobaria. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia via HIF-1alpha, and these two molecules are colocalized with presumptive regions of hypoxia. VEGF up-regulation in embryonic and fetal hearts correlates with vascular tube formation which progresses from an epicardial to endocardial direction prior to the establishment of a functional coronary circulation. Our studies on explanted embryonic quail hearts indicate that vascular tube formation is enhanced by hypoxia (5-10% O2) and inhibited by hyperoxia. Three splice variants of VEGF (122, 126, 190) were found to increase and decrease with hypoxia and hyperoxia, respectively. While VEGF synthesis is stimulated by hypoxia, there are differences in the vascular patterning between exogenous VEGF-induced vascularization and that induced by hypoxia. Thus, other, yet to be identified, molecules are recruited by hypoxia. Acute hypoxia selectively enhances at least three splice variants of VEGF-A, and also selectively up-regulates VEGFR-1 (flt-1). However, we suggest that VEGF-B, a ligand for VEGFR-1 may contribute to embryonic myocardial vascularization, since we have shown that it plays a key role in this process under normoxic conditions. A second mechanism by which hypoxia may play a role in vascularization of the heart is via its vasodilatory effects, once the coronary circulation is functional. Increased blood flow serves as a mechanical (stretch) trigger for activation of VEGF and its receptors. In sum, there is evidence that a relative hypoxia provides both metabolic and mechanical stimuli for vascular growth in the developing heart.