He Wang, Hassan Albadawi, Zakir Siddiquee, Jillian M. Stone, Mikhail P

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Altered vascular activation due to deficiency of the NADPH oxidase component p22phox He Wang, Hassan Albadawi, Zakir Siddiquee, Jillian M. Stone, Mikhail P. Panchenko, Michael T. Watkins, James R. Stone Cardiovascular Pathology Volume 23, Issue 1, Pages 35-42 (January 2014) DOI: 10.1016/j.carpath.2013.08.003 Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 1 Intimal hyperplasia in the proximal and mid carotid artery following ligation. Left common carotid artery after ligation in wild-type (A) and p22phox-deficient (B) mice. Scale bars represent 20 μm. (C) The wild-type mice were relatively resistant to the formation of intimal hyperplasia with no effect due to p22phox deficiency. n=6–8 male mice per group. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 2 Elastic fiber loss at the site of ligation in the carotid arteries. Sites of ligation in the left common carotid artery in wild-type mice (A) showing marked loss of vascular elastic fibers. In contrast, the p22phox-deficient mice (B) show preservation of the vascular elastic fibers. (C) Quantitation of vascular elastic fiber loss. *P=.005, n=5 to 6 male mice per group. At higher magnification, the vascular smooth muscle cells show disorganization in the wild-type animals at the sites of elastic fiber loss (D) compared with the smooth muscle cells in the p22phox-deficient mice (E). Scale bars represent 40 μm (A, B) or 20 μm (D, E). Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 3 Changes in the expression of MMP12, MMP3, and TIMP1. One week following carotid artery ligation, mRNA levels were assessed in full-length ligated and contralateral nonligated carotid arteries of both male and female mice by real-time PCR. (A) MMP12, (B) MMP3, (C) TIMP1, (D) MMP12/TIMP1 ratio. #P<.05 versus the nonligated side, *P<.01 versus the nonligated side, **P<.01 versus the nonligated side, and P<.01 versus the ligated p22phox-deficient group. Two-way ANOVA/Tukey, n=4 to 6 mice per group. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 4 Adventitial inflammation at the site of ligation in the carotid arteries. Shown are immunohistochemical stains for the neutrophil marker Ly-6B (A–D) and the macrophage marker F4/80 (F–I). The carotid arteries from the nonligated side show no significant inflammatory infiltrate in either wild-type (A, F) or p22phox-deficient mice (C, H). In contrast, at the site of ligation, both wild-type mice (B, G) and p22phox-deficient mice (D, I) display an inflammatory infiltrate in the adventitia of the carotid arteries. Scale bars represent 20 μm. Insets depict negative controls. (E) Quantitation of neutrophils. (J) Quantitation of macrophages. *P<.01 versus the nonligated side, two-way ANOVA/Tukey, n=3 to 4 male mice per group. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 5 Oxidative stress in the distal ligated carotid arteries. Immunohistochemical stains for the oxidative stress marker HNE in the distal ligated carotid arteries of wild-type (A) and p22phox-deficient (B) mice, with corresponding negative controls (C, D). Scale bars represent 20 μm. (E) Quantitation of the percentage of smooth muscle cell staining for HNE. *P=.008, n=3 male mice per group. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 6 Involvement of the CK1αLS/hnRNP-C pathway. In the distal carotid artery just proximal to the site of ligation, the wild-type mice (A) show strong expression of the vascular activation marker hnRNP-C in smooth muscle cells by immunohistochemistry. The p22phox-deficient mice (B) show reduced expression of hnRNP-C. Scale bars represent 20 μm. Insets depict negative controls. (C) Quantitation of vascular hnRNP-C expression. *P=.005, n=4 male mice per group. (D) Assessment of the relative MMP12/TIMP1 mRNA ratios in paired human coronary artery smooth muscle cell preparations with and without knockdown of CK1αLS. *P<.05, n=3. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 7 hnRNP-C expression in other tissues. Both wild-type (A, C, E) and p22phox-deficient (B, D, F) male mice show strong expression of hnRNP-C in the liver (A, B), heart (C, D), and periaortic soft tissue (E, F right side) by immunohistochemistry. Neither group shows strong expression of hnRNP-C in the aorta (E, F left side). Scale bars represent 20 μm. Insets depict negative controls. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Fig. 8 Vascular hnRNP-C expression in human patients with and without p22phox deficiency. Shown are histologic images of lung biopsies from a p22phox-deficient patient (A, B) and two control patients (C, D and E, F). Hematoxylin & eosin stained sections (A, C, E) show small arteries within the biopsies. By immunohistochemistry for hnRNP-C, the p22phox-deficient patient shows strong expression of hnRNP-C in periarterial cells but not in the vascular smooth muscle cells (B). In contrast, the two control patients show strong expression for hnRNP-C in both periarterial cells and in vascular smooth muscle cells (D, F). Scale bars represent 40 μm. Insets depict negative controls. (G) Quantitation of vascular smooth muscle cell hnRNP-C staining. *P<.0001, one-way ANOVA/Bonferroni, n=5 arteries per patient. Cardiovascular Pathology 2014 23, 35-42DOI: (10.1016/j.carpath.2013.08.003) Copyright © 2014 Elsevier Inc. Terms and Conditions