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.

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-β1 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 TβRIII (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-β1.

Previously, we have shown that heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) is a transforming growth factor-β1 (TGF-β1)-responsive gene in normal human lung fibroblasts and functions as a negative feedback regulator of TGF-β1 and that TGF-β1 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-β1 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-β1 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-β1 response with early enhanced Smad activation, but eventually reduced TGF-β1 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-β1 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-β1 signaling in type II AECs.

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. 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 GABA_A 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. 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.

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.