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. 2020 Mar 24;7:78.
doi: 10.3389/fmed.2020.00078. eCollection 2020.

Uremic Vascular Calcification Is Correlated With Oxidative Elastic Lamina Injury, Contractile Smooth Muscle Cell Loss, Osteogenesis, and Apoptosis: The Human Pathobiological Evidence

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Uremic Vascular Calcification Is Correlated With Oxidative Elastic Lamina Injury, Contractile Smooth Muscle Cell Loss, Osteogenesis, and Apoptosis: The Human Pathobiological Evidence

Jia-Feng Chang et al. Front Med (Lausanne). .
Free PMC article

Abstract

Background: Uremic vascular calcification (UVC) is reminiscent of osteogenesis and apoptosis in vascular smooth muscle cell (VSMC). We aimed to identify how circulating procalcific particles dramatically leak into VSMC layer in human tissue models of vascular rings. Methods: According to baseline estimated glomerular filtration rate (eGFR), patients following lower extremity amputation were divided into three groups: normal renal function (eGFR ≧ 60 ml/min), mild-to-moderate (15 ml/min < eGFR ≧ 60 ml/min) and severe chronic kidney disease (CKD) (eGFR ≦ 15 ml/min). Arterial specimens with immunohistochemistry stain were quantitatively analyzed for UVC, internal elastic lamina (EL) disruption, α-SMA, osteogenesis, apoptosis, and oxidative injury. Correlations among UVC severity, eGFR, EL disruption, osteogenesis, and oxidative injury were investigated. Results: CKD arteries were associated with eGFR-dependent EL disruption corresponding to UVC severity. CKD arteries exhibited lower α-SMA, higher expressions of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), indicative of contractile VSMC loss, and apoptosis. Enhanced expressions of alkaline phosphatase and Runx2 were presented in VSMCs of CKD arteries, indicative of osteogenic differentiation. Above eGFR-dependent UVC and EL disruption correlated expressions of 8-hydroxy-2'-deoxyguanosine (8-OHdG), indicating oxidative EL injury promoted procalcific processes. Conclusions: Circulating uremic milieu triggers vascular oxidative stress, leading to progressive internal EL disruption as a key event in disabling VSMC defense mechanisms and catastrophic mineral ion influx into VSMC layer. Oxidative EL injury begins in early CKD, corresponding with active VSMC re-programming, apoptosis, and ultimately irremediable UVC. In light of this, therapeutic strategies targeting oxidative tissue injury might be of vital importance to hinder the progression of UVC related cardiovascular events.

Keywords: apoptosis; contractile smooth muscle cell; elastic lamina; osteogenesis; oxidative injury; uremic vascular calcification.

Figures

Figure 1
Figure 1
Effects of eGFR-dependent internal EL loss on UVC in CKD arteries. Muscular arteries were classified into three study groups of normal controls, mild-to-moderate and severe CKD. Control groups, eGFR ≧ 60 ml/min; Mild-to-moderate CKD, 15 ml/min < eGFR ≦ 60 ml/min; Severe CKD, eGFR ≦ 15 ml/min. (A) Internal EL defect areas were examined using EVG stain. Yellow arrows indicated internal EL loss in mild-to-moderate and severe CKD groups, compared to normal controls. Scale bars in the upper panels are 200 μm and 40 μm in the lower panels. (B) Calcium deposits were localized using Von Kossa stain. Note that stretching, fragmentation and disruption of internal EL were presented adjacent to the eGFR-dependent UVC regions. Fifty-one human samples were used for the experiments: controls (n = 19); mild-to-moderate CKD (n = 15); severe CKD (n = 17). For image analyses, three random high-resolution fields were obtained in each sample. Quantitative analyses of EVG and Von Kossa staining were performed using ImageJ software. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001. A, adventitia; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; EL, elastic lamina; EVG, elastic tissue fibers-Verhoeff's Van Gieson; I, intima; M, media; UVC, uremic vascular calcification.
Figure 2
Figure 2
Effects of eGFR-dependent UVC on VSMC contractility impairment and apoptosis in tunica media. Quantitative analysis of immunohistochemical staining for (A) α-SMA (B) Caspase-3 and (C) TUNEL was performed using ImageJ software. Note that the decrease of contractile VSMCs and VSMC apoptosis were presented adjacent to the eGFR-dependent UVC regions. Fifty-one human samples were used for the experiments: controls (n = 19); mild-to-moderate CKD (n = 15); severe CKD (n = 17). For image analyses, three random high-resolution fields were obtained in each sample. Scale bars in the upper panels are 200 and 40 μm in the lower panels. Data are expressed as mean ± SD. **P < 0.01; ***P < 0.001. A, adventitia; eGFR, estimated glomerular filtration rate; I, intima; M, media; SMA, smooth muscle actin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UVC, uremic vascular calcification; VSMC, vascular smooth muscle cells.
Figure 3
Figure 3
Effects of eGFR-dependent osteogenic transcription factor Runx2 on ALP expressions. Quantitative analysis of immunohistochemical staining for (A) ALP and (B) Runx2 was performed using ImageJ software. Yellow arrows indicate the positive staining of ALP in VSMCs. Fifty-one human samples were used for the experiments: controls (n = 19); mild-to-moderate CKD (n = 15); severe CKD (n = 17). For image analyses, three random high-resolution fields were obtained in each sample. Scale bars in the upper panels are 200 and (A) 40 μm and (B) 120 μm in the lower panels. Note that the positive staining areas of ALP and Runx2 were compatible. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. A, adventitia; ALP, alkaline phosphatase; eGFR, estimated glomerular filtration rate; I, intima; M, media; Runx2, runt-related transcription factor 2.
Figure 4
Figure 4
Effects of eGFR-dependent UVC on oxidative DNA damages of VSMCs. (A) Vascular oxidative injury was examined by immunohistochemical staining of 8-OHdG. Yellow arrows indicate positive staining of 8-OHdG in nuclei of VSMCs. (B) Calcium deposits were localized using Von Kossa stain. Scale bars in the upper panels are 200 μm, and (A) 120 μm and (B) 40 μm in the lower panels. Fifty-one human samples were used for the experiments: controls (n = 19); mild-to-moderate CKD (n = 15); severe CKD (n = 17). For image analyses, three random high-resolution fields were obtained in each sample. Quantitative analysis for positive staining was performed using ImageJ software. Data are expressed as mean ± SD. ***P < 0.001. 8-OHdG, 8-hydroxy-2'-deoxyguanosine; A, adventitia; eGFR, estimated glomerular filtration rate; I, intima; M, media; UVC, uremic vascular calcification; VSMC, vascular smooth muscle cell.
Figure 5
Figure 5
The correlation analysis between UVC, eGFR, internal EL disruption, osteogenic differentiation, and oxidative damage. (A) The correlation coefficient r between vascular calcification area and eGFR is−0.396 (P < 0.01). (B) The correlation coefficient r between vascular calcification area and EVG loss area is 0.455 (P < 0.01). (C) The correlation coefficient r between vascular calcification area and ALP expression is 0.702 (P < 0.01). (D) The correlation coefficient r between vascular calcification area and 8-OHdG positive cells is 0.638 (P < 0.01). Fifty-one human samples were used for the experiments. Quantitative analysis for positive staining was performed using ImageJ software. 8-OHdG, 8-hydroxy-2′-deoxyguanosine; eGFR, ALP, alkaline phosphatase; estimated glomerular filtration rate; EVG, elastic tissue fibers—Verhoeff's Van Gieson stain.
Figure 6
Figure 6
A comprehensive pathobiological insight into CKD-driven UVC. The direct exposure to circulating uremic milieu triggers vascular oxidative injury, leading to EL disruption as the key event in disabling VSMC defense mechanisms. Loss of intima integrity might be the rate-determining step in early stage of CKD, corresponding with oxidative VSMC re-programming, apoptosis, and ultimately irremediable UVC. Oxidative EL injury induces catastrophic mineral ion influx into VSMC layer, thus novel antioxidants will be of vital importance in treating UVC related cardiovascular events. ALP, alkaline phosphatase; Ca, calcium cations; CKD, chronic kidney disease; ECM, extracellular matrix; EL, elastic lamina; P, phosphate anions; TA, tunica adventitia; TI, tunica intima; TM, tunica media; ROS, reactive oxygen species; Runx2, runt-related transcription factor 2; UVC, uremic vascular calcification; VSMC, vascular smooth muscle cell. 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

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