Inactivation of Semicarbazide-Sensitive Amine Oxidase Stabilizes the Established Atherosclerotic Lesions via Inducing the Phenotypic Switch of Smooth Muscle Cells

<div><p>Given that the elevated serum semicarbazide-sensitive amine oxidase (SSAO) activity is associated with the severity of carotid atherosclerosis in clinic, the current study aims to investigate whether SSAO inactivation by semicarbazide is beneficial for established atherosclerotic lesions in LDLr knockout mice on a high-fat/high- cholesterol Western-type diet or after dietary lipid lowering. Despite no impact on plasma total cholesterol levels, the infiltration of circulating monocytes into peripheral tissues, and the size of atherosclerotic lesions, abrogation of SSAO activity resulted in the stabilization of established lesions as evidenced by the increased collagen contents under both conditions. Moreover, SSAO inactivation decreased Ly6C^high monocytosis and lesion macrophage contents in hypercholesterolemic mice, while no effect was observed in mice after normalization of hypercholesterolemia by dietary lipid lowering. Strikingly, abrogation of SSAO activity significantly increased not only the absolute numbers of smooth muscle cells (SMCs), but also the percent of SMCs with a synthetic phenotype in established lesions of mice regardless of plasma cholesterol levels. Overall, our data indicate that SSAO inactivation <i>in vivo</i> stabilizes the established plaques mainly <i>via</i> inducing the switch of SMCs from a contractile to a synthetic phenotype. Targeting SSAO activity thus may represent a potential treatment for patients with atherosclerosis.</p></div

SSAO inactivation stabilized the established atherosclerotic lesions under hypercholesterolemia.

<p>Female LDLr KO mice were fed WTD for 6 weeks to induce the formation of atherosclerotic lesions. Thereafter, 0.125% semicarbazide were added in drinking water to inactivate SSAO during the subsequent 3 weeks (A) before the analysis of SSAO activities in the visceral adipose tissue (B), plasma cholesterol levels (C) and atherosclerotic lesions at the aortic root (D-G). (D) A scattered dot plot (<i>left</i>) or representative photomicrographs showing the size of atherosclerotic lesions. Sections were stained with oil-red-O (original magnification 40x, <i>right panel</i>). Each dot represents the mean lesion area of a single mouse, and the horizontal bar indicates the mean value of the group (<i>left panel</i>). (E) Representative photomicrographs showing macrophages and collagens in lesions. Sections of aortic roots were stained with antibodies against Moma-2 to visualize macrophages (brown, 100x) or with Masson’s Trichrome Accustain to visualize collagens (blue, 100x). (F & G) Bar graphs showing the lesion contents of macrophages (F), collagens (G), necrotic core (H) and cap thickness (I). Results were expressed as mean±SEM. Statistically significant difference *p<0.05, **p<0.01, and ***p<0.001 <i>vs</i> vehicle.</p

SSAO inactivation increased the percent of synthetic SMCs <i>in</i> established lesions under hypercholesterolemia.

<p>Female LDLr KO mice were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152758#pone.0152758.g001" target="_blank">Fig 1</a>. (A) A bar graph (<i>left</i>) and representative photomicrographs (<i>right</i>) showing the lesion contents of SMCs. (B) A bar graph (<i>left</i>) or representative photomicrographs (<i>right</i>) showing the lesion contents of SMCs positive for Ki67. Sections of the aortic root were stained with the antibody against α-actin in the absence (A, purple for α-actin, 100x) or presence of anti-Ki67 (B, brown for α-actin while nuclear dark blue for Ki67, indicated by arrows, 400x) to visualize total or proliferative SMCs, respectively. Nuclei were counterstained by fast nuclear red (nuclear pink, B). (C) Dot plots showing proliferative VSMCs. Aortic cells were stained with antibodies against CD45, α-actin, and Ki67. The percent of proliferative (Ki67⁺) SMCs among α-actin⁺CD45^- aortic cells are analyzed. (D) Bar graph showing the percent of proliferative (Ki67⁺) SMCs. Results were expressed as mean±SEM. Statistically significant difference *p<0.05 <i>vs</i> vehicle.</p

Effect of SSAO inactivation on the migration of monocytes under hypercholesterolemia.

<p>Female LDLr KO mice were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152758#pone.0152758.g001" target="_blank">Fig 1</a>. Upon sacrifice, the percentage of CD11b⁺Ly6G^- (A) and CD11b⁺Ly6G^-Ly6C^high monocytes (B) in the blood, peritoneal cavity, and spleen were analyzed by flow cytometry. Comparable absolute numbers of total cells were obtained in corresponding tissues of mice from each group. Results were expressed as mean±SEM. Statistically significant difference *p<0.05 and ***p<0.001 <i>vs</i> vehicle.</p

Soluble SSAO inhibited the phenotype switch of VSMCs <i>in vitro</i>.

<p>Murine VSMCs were starved and stimulated with 20 ng/mL murine PDGF-BB to induce a phenotype switch in the absence/presence of soluble bovine SSAO and methylamine together with/without semicarbazide for 24 hours. (A) Representative pictures of VSMC migration through scratch-wound. (B) Bar graph showing the quantification of migrated cell numbers under field of view. The average number of migrated cells was determined by counting 6 fields of view per well. (C) Bar graph showing the percent of EdU incorporated proliferative VSMCs determined by flow cytometric analysis. (D) Bar graph showing collagen productions of VSMCs. Values represent the mean ±SEM (n = 4 wells). Statistically significant difference ***p<0.001 and **p<0.01 <i>vs</i> medium.</p

SSAO inactivation increased the synthetic SMCs in established lesions under normacholesterolemia after dietary lipid lowering.

<p>Male LDLr KO mice were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152758#pone.0152758.g004" target="_blank">Fig 4</a>. (A) A bar graph (<i>left</i>) or representative photomicrographs (<i>right</i>) showing the lesion contents of SMCs. (B) A bar graph (<i>left</i>) or representative photomicrographs (<i>right</i>) showing the lesion contents of SMCs positive for Ki67 in vehicle- or semicarbazide-treated mice. Sections of the aortic root were stained with the antibody against α-actin in the absence (A, purple for α-actin, 100x) or presence of anti-Ki67 (B, red for α-actin while nuclear dark blue for Ki67, indicated by arrows, 400x) to visualize total or proliferative SMCs, respectively. Nuclei were counterstained by fast nuclear red (nuclear pink, B). (C) Dot plots showing proliferative VSMCs. Aortic cells were stained with antibodies against CD45, α-actin, and Ki67. The percent of proliferative (Ki67⁺) SMCs among α-actin⁺CD45^- aortic cells are analyzed. (D) Bar graph showing the percent of proliferative (Ki67⁺) SMCs. Results were expressed as mean±SEM. Statistically significant difference *p<0.05 <i>vs</i> vehicle.</p

SSAO inactivation stabilized the established atherosclerotic lesions after normalization of hypercholesterolemia by diet lipid lowering.

<p>Male LDLr KO mice were fed WTD for 9 weeks to induce the formation of atherosclerotic lesions. Thereafter, these animals were fed regular chow diet to normalize hypercholesterolemia (A). Drinking water containing 0.125% semicarbazide was given to these chow-fed animals in the subsequent 6 weeks (A) before the analysis of SSAO activities in the visceral adipose tissue (B), plasma cholesterol levels (C) or atherosclerotic lesions at the aortic root (D-G). (D) A scattered dot plot (<i>left</i>) or representative photomicrographs showing the size of atherosclerotic lesions. Sections were stained with oil-red-O (original magnification 40x, <i>right panel</i>). Each dot represents the mean lesion area of a single mouse, and the horizontal bar indicates the mean value of the group (<i>left panel</i>). (E) Representative photomicrographs showing the lesion contents of Moma-2⁺ macrophages (<i>upper panel</i>) or collagens (<i>lower panel</i>) in each group. Sections of aortic roots were stained with antibodies against Moma-2 to visualize macrophages (brown, 100x) or with Masson’s Trichrome Accustain to visualize collagens (blue, 100x). (F & G) Bar graphs showing the lesion contents of macrophages (F), collagens (G), necrotic core (H), and cap thickness (I) in vehicle- or semicarbazide-treated mice. Results were expressed as mean ±SEM. Statistically significant difference ***p<0.001 and *p<0.05 <i>vs</i> vehicle.</p

IC₅₀ of indole alkaloid against lipase [23].

Regular use of Thai kratom has been linked to reduced blood triglyceride levels and body mass index (BMI) in healthy individuals. We analyzed Green Thai Kratom (GTK) and Red Thai Kratom (RTK) to investigate their effects on pancreatic digestive enzymes. The ethanol extracts of GTK and RTK inhibited lipase activity more strongly than alpha-glucosidase activity, suggesting the presence of lipase inhibitors. Mitragynine, the major compound in GTK, showed potent lipase inhibition and moderate alpha-glucosidase inhibition. Quercetin, found in both extracts, strongly inhibited alpha-glucosidase but had limited effects on lipase. These findings suggest that mitragynine and quercetin may hinder triglyceride and starch digestion. Combination inhibition studies revealed synergistic effects between mitragynine and quercetin on alpha-glucosidase activity. Additionally, both GTK and RTK extracts reduced fat accumulation in 3T3-L1 adipocyte cells, with quercetin specifically inhibiting Acetyl-CoA carboxylase 1 (ACC1), a key enzyme in fatty acid biosynthesis. Thus, GTK and RTK extracts, particularly mitragynine and quercetin, exhibit potential anti-obesity effects. We report the novel finding that Thai kratom inhibits de novo fatty acid synthesis by targeting ACC1, resulting in decreased fat accumulation in adipocytes. Regular use of Thai kratom in specific populations may improve blood triglyceride levels and reduce BMI by inhibiting lipase, alpha-glucosidase, and ACC1 activity. Further clinical trials are needed to determine optimal dosage, duration, toxicity levels, and potential side effects of Kratom use.</div

Effect of Red and Green Kratom on fat accumulation in 3T3-L1 cells.

After 72 hours, lipid accumulation in 3T3-L1 cells treated with Red and Green Kratom (0–10 μg/mL) was determined using Oil Red O staining (A). Lipid accumulation at each concentration was assessed in triplicate experiments and determined by measuring the absorbance of Oil Red O at 500 nm (B). The asterisk (*) indicates a statistically significant decrease in fat accumulation compared to 0 μM of each test inhibitor (p < 0.05, Student’s t-test). The experiments were done in triplicate.</p

Inhibitory effect of mitragynine and quercetin against ACC1-medated Accetyl-CoA.

Inhibitory effect of mitragynine and quercetin against ACC1-medated Accetyl-CoA.</p