Cathepsin S Deficiency Mitigated Chronic Stress-Related Neointimal Hyperplasia in Mice

Abstract

Background Exposure to chronic psychosocial stress is a risk factor for atherosclerosis-based cardiovascular disease. We previously demonstrated the increased expressions of cathepsin S (CatS) in atherosclerotic lesions. Whether CatS participates directly in stress-related neointimal hyperplasia has been unknown. Methods and Results Male wild-type and CatS-deficient mice that underwent carotid ligation injury were subjected to chronic immobilization stress for morphological and biochemical studies at specific times. On day 14 after stress/surgery, stress enhanced the neointima formation. At the early time points, the stressed mice had increased plaque elastin disruption, cell proliferation, macrophage accumulation, mRNA and/or protein levels of vascular cell adhesion molecule-1, angiotensin II type 1 receptor, monocyte chemoattractant protein-1, gp91^phox, stromal cell-derived factor-1, C-X-C chemokine receptor-4, toll-like receptor-2, toll-like receptor-4, SC 35, galectin-3, and CatS as well as targeted intracellular proliferating-related molecules (mammalian target of rapamycin, phosphorylated protein kinase B, and p-glycogen synthase kinase-3α/β). Stress also increased the plaque matrix metalloproteinase-9 and matrix metalloproteinase-2 mRNA expressions and activities and aorta-derived smooth muscle cell migration and proliferation. The genetic or pharmacological inhibition of CatS by its specific inhibitor (Z- FL -COCHO) ameliorated the stressed arterial targeted molecular and morphological changes and stressed aorta-derived smooth muscle cell migration. Both the genetic and pharmacological interventions had no effect on increased blood pressure in stressed mice. Conclusions These results demonstrate an essential role of CatS in chronic stress-related neointimal hyperplasia in response to injury, possibly via the reduction of toll-like receptor-2/toll-like receptor-4-mediated inflammation, immune action, and smooth muscle cell proliferation, suggesting that CatS will be a novel therapeutic target for stress-related atherosclerosis-based cardiovascular disease.

Keywords: hyperplasia; hypertension; protease; stress; vascular disease.

Figure 1

Chronic stress accelerated injury‐related neointimal formation in cathepsin S wild‐type (CatS^+/+) mice. A, Schematic diagram of mouse right carotid ligation surgery and sampling procedures at the indicated time points. Dihydroethidium was used for oxidative stress staining. B, Representative hematoxylin and eosin (HE) staining images of right and left carotid arteries of the nonstressed and stressed groups. C and D, Quantitative data showing the neointimal areas and the ratio of neointima area/media area in injured arteries of both experimental groups. Bar=100 μm. Results are mean±SEM (n=7). EVG indicates Elastica van Gieson (for elastin staining); NS, no significance; PCNA, proliferating cell nuclear antigen (for proliferation staining); PSR, picrosirius red (for collagen staining); RT‐PCR, real‐time polymerase reaction chain. ***P<0.01 vs nonstressed group by Student t test.

Figure 2

Stress accelerates cell proliferation and media elastic laminal degradation and collagen in cathepsin S wild‐type (CatS^+/+) mice. A and B, Representative proliferating cell nuclear antigen (PCNA) immunostaining images of media smooth muscle cell proliferation and combined quantitative data for PCNA ⁺ cells. C through F, Representative images and quantitative data for the number of broken elastin fragments (Elastica van Gieson) and collagen content (picrosirius red [PSR]). Bar=100 μm. The results are mean±SEM (n=5–6). ***P<0.001 vs CatS^+/+ group by Student t test.

Figure 3

Stress accelerates cathepsin S wild‐type (CatS^+/+) aorta‐derived smooth muscle cell migration. A, Representative images and quantitative data for the sprouted cell numbers and cell sprouted areas at the indicated time points. B and C, Quantitative data showing the sprouted cell numbers and cell sprouted areas of the 2 experimental groups at the indicated time points. Bar=100 μm. Results are mean±SEM (n=5). ***P<0.001 vs corresponding nonstressed groups by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 4

Stress produced a harmful change in the targeted oxidative stress–, inflammation‐, and proteolysis‐related gene expressions in the carotid arteries of cathepsin S wild‐type (CatS^+/+) mice. A through F, Quantitative polymerase chain reaction data show the levels of p22^phox, p47^phox, p67^phox, nicotinamide‐adenine dinucleotide phosphate, reduced form, oxidase 1 (NOX‐1), gp91^phox, intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), monocyte chemoattractant protein‐1 (MCP‐1), stromal cell–derived factor‐1α (SDF‐1α), C‐X‐C chemokine receptor‐4 (CXCR‐4), toll‐like receptor (TLR)‐2, TLR‐4, tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), angiotensin II receptor 1α (ATR1α), matrix metalloproteinase (MMP)‐9, MMP‐2, cathepsin L (CatL), and CatS mRNAs. Results are mean±SEM (n=5–7). ***P<0.001 vs nonstressed group by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 5

Immunostaining showed the cell sources of cathepsin S (CatS) in injured arteries. A and B, Representative images of CatS staining that show the positive staining signaling was pronounced in the media and neointima (thin) of injured vessels from wild‐type CatS (CatS^+/+) mice on day 4 after surgery, whereas no signaling was detected in the whole arterial walls of CatS‐deficient (CatS^−/−) mice. C, CatS staining of cross‐sections taken on day 4 after ex vivo cultured aortae of CatS^+/+ mice received stress. D and E, Representative images and quantitative data show the gp91^phox+ staining area. Results are mean±SEM (n=5–7). NS indicates no significance. ***P<0.001 vs nonstressed group by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 6

Stress accelerated the oxidative stress production and macrophage infiltration in response to injury at day 4. A and B, Representative images of dihydroethidium staining and quantitative data for positive staining area of the injured arterial tissues. C and D, Representative images and quantitative data for CD68⁺ cell numbers (arrowheads). E through G, Representative images of gelatin zymography and combined quantitative data for gelatinolytic activities of matrix metalloproteinase (MMP)‐2 and MMP‐9 in the nonstressed and stressed groups. Bar=100 μm. Results are mean±SEM (n=4–6). CatS^+/+ indicates cathepsin S wild type; NS, no significance. ***P<0.001 vs nonstressed group by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 7

Stress altered the levels of targeted protein in the injured arteries. A through C, Representative images and combined quantitative data for the levels of targeted proteins. Results are mean±SEM (n=5–7). AKT indicates protein kinase B; ATR1, angiotensin II receptor 1; CatS^+/+, cathepsin S wild type; Erk, extracellular signal‐regulated kinase; GSK, phosphoglycogene synthesis kinase; mTOR, mammalian target of rapamycin; NS, no significance; p‐AKT, phosphorylated AKT; p‐Erk, phosphorylated Erk; p‐mTOR, phosphorylated mTOR. ***P<0.001 vs the relative controls by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 8

Cathepsin S deficiency (CatS^−/−) alleviated injury‐induced neointimal formation in the mice subjected to chronic stress. A, Representative hematoxylin and eosin staining images of right and left carotid arteries of wild‐type (CatS^+/+) and CatS^−/− stressed mice. B and C, Quantitative data showing the neointimal areas and the ratio of neointima area/media area in injured arteries of the 2 experimental groups. Bar=100 μm. The results are mean±SEM (n=5–7). NS indicates no significance. ***P<0.01 vs CatS^+/+ group by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 9

Cathepsin S deficiency (CatS^−/−) mitigated cell proliferation and elastin degradation. A and B, Representative proliferating cell nuclear antigen (PCNA) immunostaining images of media smooth muscle cell proliferation and combined quantitative data for PCNA ⁺ cells. C through F, Representative images and quantitative data for the number of broken elastin fragments (Elastica van Gieson [EVG]) and collagen content (picrosirius red [PSR]). Bar=100 μm. Results are mean±SEM (n=5–6). ***P<0.001 vs cathepsin S wild type (CatS^+/+) by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 10

Cathepsin S deficiency (CatS^−/−) impaired aorta‐derived smooth muscle migration in the stressed mice. A through C, Representative images and quantitative data showing the sprouted cell numbers and sprouted areas. D through F, Representative images and quantitative data showed that CatS^−/− reduced the neointimal areas and the ratio of neointima area/media area in injured arteries of the mice even without chronic stress. Bar=100 μm. The results are mean±SEM (n=6–7). CatS^+/+ indicates cathepsin S wild type; HE, hematoxylin and eosin; NS, no significance. ***P<0.001 vs corresponding control group by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 11

Cathepsin S deficiency (CatS^−/−) mitigated the expressions of targeted oxidative stress–, inflammation‐, and proteolysis‐related genes in the carotid arteries of the stressed mice at day 4 after surgery. A through F, Quantitative polymerase chain reaction data show the levels of p22^phox, gp91^phox, p47^phox, p67^phox, nicotinamide‐adenine dinucleotide phosphate, reduced form, oxidase 1 (NOX‐1), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), monocyte chemoattractant protein‐1 (MCP‐1), stromal cell–derived factor‐1α (SDF‐1α), C‐X‐C chemokine receptor‐4 (CXCR‐4), toll‐like receptor (TLR)‐2, TLR‐4, tumor necrosis factor (TNF)‐α, interleukin‐1β (IL‐1β), angiotensin II receptor 1α (ATR1α), matrix metalloproteinase (MMP)‐9, MMP‐2, cathepsin L (CatL), and CatS mRNAs. Results are mean±SEM (n=5–7). CatS^+/+ indicates CatS wild type. ***P<0.001 vs corresponding CatS^+/+ by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 12

Cathepsin S deficiency (CatS^−/−) reduced the oxidative stress production and macrophage infiltration in the injured arteries of stressed mice at day 4 after surgery. A and B, Representative images and quantitative data for the dihydroethidium staining area of the injured carotid arteries of stressed CatS wild type (CatS^+/+) and CatS^−/− mice. C and D, Representative images and quantitative data of the numbers of CD68⁺ macrophages. E through G, Representative images of gelatin zymography and combined quantitative data for the gelatinolytic activities of matrix metalloproteinase (MMP)‐2 and MMP‐9 in the carotid arteries of stressed CatS^+/+ and CatS^−/− mice. Bar=100 μm. Results are mean±SEM (n=4–6). NS indicates no significance. ***P<0.001 vs corresponding CatS^+/+ by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 13

Cathepsin S (CatS) deletion improved targeted molecule changes in the injured arteries of the stressed mice. A through C, Representative images and combined quantitative data for the levels of targeted molecule proteins. The results are mean±SEM (n=5–7). AKT indicates protein kinase B; ATR1, angiotensin II receptor 1; CatS^+/+, CatS wild type; CatS^−/−, CatS deficient; Erk, extracellular signal‐regulated kinase; GSK, phosphoglycogene synthesis kinase; mTOR, mammalian target of rapamycin; NS, no significance; p‐AKT, phosphorylated AKT; p‐Erk, phosphorylated Erk; p‐GSK, phosphorylated GSK; p‐mTOR, phosphorylated mTOR. ***P<0.001 vs CatS^+/+ by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 14

Cathepsin S inhibitor (CatS‐I) alleviated injury‐induced neointimal formation at day 14 after surgery. A, Representative hematoxylin and eosin staining images of the right and left carotid arteries of stressed CatS wild type (CatS^+/+) mice treated with CatS‐I (CatS‐I [+]) or without CatS‐I (CatS‐I [−]). B and C, Quantitative data showing the neointimal areas and the ratio of neointima area/media area in injured arteries of the 2 experimental groups. Bar=100 μm. Results are mean±SEM (n=7). NS indicates no significance. ***P<0.001 vs CatS‐I (−) group by Student t test.

Figure 15

Cathepsin S inhibition (CatS‐I) impaired the aorta‐derived smooth muscle cell migration in stressed CatS wild‐type (CatS^+/+) mice at day 7 after surgery. A through C, Representative images and quantitative data for sprouted cell numbers and sprouted areas at day 7 after culture. D through F, Representative images and quantitative data revealed that CatS‐I also suppressed the neointimal areas and the ratio of neointima area/media area in injured arteries of the mice without stress. Bar=100 μm. Results are mean±SEM (n=6–7). HE indicates hematoxylin and eosin; NS, no significance. ***P<0.001 vs without CatS‐I by Student t test.

Figure 16

Cathepsin S inhibition (CatS‐I) mitigated the expressions of targeted oxidative stress–, inflammation‐, and proteolysis‐related genes in the carotid arteries of the stressed CatS wild‐type (CatS^+/+) mice at day 4 after surgery. A through F, Quantitative polymerase chain reaction data show the levels of p22^phox, gp91^phox, p47^phox, p67^phox, nicotinamide‐adenine dinucleotide phosphate, reduced form, oxidase 1 (NOX‐1), intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), monocyte chemoattractant protein‐1 (MCP‐1), stromal cell–derived factor‐1α (SDF‐1α), C‐X‐C chemokine receptor‐4 (CXCR‐4), toll‐like receptor (TLR)‐2, TLR‐4, tumor necrosis factor (TNF)‐α, interleukin‐1β (IL‐1β), angiotensin II receptor 1α (ATR1α), matrix metalloproteinase (MMP)‐9, MMP‐2, cathepsin (CatL), and CatS mRNAs of mice with CatS‐I (CatS‐I [−]) and mice with CatS‐I (CatS‐I [+]). Results are mean±SEM (n=5–7). NS indicates no significance. **P<0.01/***P<0.001 vs corresponding CatS‐I (−) mice by 1‐way ANOVA, followed by Tukey post hoc tests.

Figure 17

Cathepsin S inhibitor (CatS‐I) mitigated oxidative stress production and reduced the macrophage infiltration in injured arteries of stressed CatS wild‐type (CatS^+/+) mice at day 4 after injury. A and B, Representative images and quantitative data for the dihydroethidium staining area of the injured carotid arteries of stressed mice without CatS‐I (CatS‐I [−]) and mice with CatS‐I (CatS‐I [+]). C and D, Representative images and quantitative data of the numbers of CD68⁺ macrophages. E through G, Representative images of gelatin zymography and combined quantitative data for the gelatinolytic activities of matrix metalloproteinase (MMP)‐2 and MMP‐9 in the carotid arteries of stressed CatS‐I (−) and CatS‐I (+) mice. Bar=100 μm. Results are mean±SEM (n=4–7). NS indicates no significance. ***P<0.001 vs corresponding CatS‐I (−) group by 1‐way ANOVA, followed by Tukey post hoc tests or Student t test.

Figure 18

Cathepsin S inhibition (CatS‐I) mitigates the stress‐related harmful targeted protein changes in the injured arteries of CatS wild‐type (CatS^+/+) mice at day 4 after injury. A through C, Representative images and quantitative data showing the levels of targeted proteins. Results are mean±SEM (n=5–7). Akt indicates protein kinase B; ATR1, angiotensin II receptor 1; CatS‐I (+), with CatS‐I; CatS‐I (−), without CatS‐I; Erk, extracellular signal‐regulated kinase; GSK, phosphoglycogene synthesis kinase; mTOR, mammalian target of rapamycin; NS, no significance; p‐Akt, phosphorylated Akt; p‐Erk, phosphorylated Erk; p‐GSK, phosphorylated GSK; p‐mTOR, phosphorylated mTOR. ***P<0.001 vs corresponding CatS‐I (−) mice by 1‐way ANOVA, followed by Tukey post hoc tests.