Fig 2: Illustration of potential mechanisms for the beneficial effects of NAC on the recovery of CLI in T2D mice. We proposed that NAC enhances the recovery of CLI in T2D mice through multiple mechanisms: (1) attenuation of excessive ROS production; (2) reduction of plasma TNF-a level; (3) decrease of insulin resistance (evidenced by increased levels of p-IRS-1 and the HOMA-IR index) in ischemic limbs; and (4) preservation of the expressions of antioxidant enzymes, phosphorylated IRS-1, Akt, and eNOS. CLI, Critical limb ischemia; T2D, type-2 diabetes; NAC, N-acetylcysteine; ROS, reactive oxygen species; SOD-1, copper-zine superoxide dismutase in cytoplasm; Gpx-1, glutathione peroxidase-1; HOMA-IR, homeostatic model assessment of insulin resistance; -, inhibit; ?, increase; ?, decrease.

Fig 3: Immunoreactivity of endothelial nitric oxide synthase (eNOS) in pulmonary arteries of normal (Nor) and Sugen5416/hypoxia/normoxia-exposed PAH rats. (A) Representative photomicrographs of eNOS-stained pulmonary arteries at the 1-, 3-, 5-, and 8-week time points after the Sugen5416 injection (upper: 50–100 µm of mediuim arteries, lower: <50 µm of smaller arteries). Scale bars indicate 50 µm. (B) Black, gray and white bars indicate immunoreactivity of eNOS in the innermost layer cells of smaller (<50 µm) and medium (50–100 µm) pulmonary arteries, respectively. The determination of immunohistochemical grade was described in method section. N = 4, each. *P < 0.05 and **P < 0.01, versus normal; † P < 0.05 and ‡ P < 0.01, versus 1-week; § P < 0.01, versus 3-week.

Fig 4: In vitro assessment of left ventricular NO bioavailability and oxidative stress in Normal and DM + HFD + CKD swine. Left ventricular myocardial nitrite (NO2-) and nitrate (NO3-) levels plotted as a function of eNOS protein levels (a), left ventricular 8-isoprostane levels (b), NADPH-oxidase 2 (NOX2) mRNA expression levels (c) left ventricular total antioxidant capacity (d), and in vitro small coronary artery endothelium-dependent vasodilation to bradykinin (BK) in the absence/presence of ROS scavengers Tempol and MPG (e and f) in Normal and DM + HFD + CKD swine. Values are mean ± SEM. *P < 0.05 and (*)P = 0.086 for DM + HFD + CKD versus Normal by logistic regression analysis (a), Student’s t test (b–d ) and two-way ANOVA for repeated measures (f)

Fig 5: LEENE homolog in mouse. a Comparison of human LEENE and mouse BY707159.1 loci. b Putative KLF2/4 TFBS in DNA region encoding BY707159.1, indicated by blue (for KLF2) and red (for KLF4). c Sequence alignment between human LEENE and BY707159.1. d Measurement of BY707159.1 RNA levels in thoracic aorta (TA) and aortic arch (AA) using qPCR. e qPCR of BY707159.1 and eNOS RNA level in isolated mouse lung ECs transfected with scramble or LEENE LNA. In each experiment, lungs from four animals were pooled for isolation and transfection. Data are average from four independent experiments. Error bars present mean ± SEM. *p < 0.05 between AA and TA in d and between scramble and LNA in e based on Student’s t test