Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ∼1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ∼50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,207 ± 24,669, N=7 versus 101,537 ± 15,881, N=20, P<0.08) despite a reduced frequency of Ca2+ cycling (events s−1 cell−1: 0.011 ± 0.004, N=8 versus 0.021 ± 0.003, N=19, P<0.05) than those without T2D. SKP-derived VSMCs from subjects with T2D also manifest enhanced sensitivity to phenylephrine (PE) in an impedance-based assay (EC50 nM: 72.3 ± 63.6, N=5 versus 3,684 ± 3,122, N=9, P<0.05), and impaired wound closure in vitro (% closure: 21.9 ± 3.6, N=4 versus 67.0 ± 10.3, N=4, P<0.05). Compared with aortic- and saphenous vein-derived primary VSMCs, SKP-derived VSMCs are functionally distinct, but mirror defects of T2D also exhibited by primary VSMCs. Conclusion: Skin biopsies from older adults yield sufficient SKPs to differentiate VSMCs, which reveal abnormal phenotypes of T2D that survive differentiation and persist even after long-term normoglycemic culture.

Introduction

Type 2 diabetes (T2D) is a complex polygenic, multifactorial disease with a large environmental component. Subjects with T2D are at increased risk of developing cardiovascular disease (CVD) as a result of their increased burden of atherosclerosis, propensity toward thrombosis, and other ill-defined factors [14]. To date, animal models fail to adequately recapitulate human aspects of the vascular dysfunction associated with T2D, including hypertension (HTN) and atherosclerosis [5,6]. Our limited knowledge of the mechanisms underlying CVD in T2D is also due, in part, to the inaccessibility of human cell types such as endothelial and vascular smooth muscle cells (VSMCs) from affected subjects.

We have previously differentiated VSMC from mouse embryonic stem cells (ESCs) [7], as well as from specific progenitor cells found during cardiovascular-directed differentiation [8]. We successfully reproduced similar approaches in human ESC and induced pluripotent stem cell (iPSC) lines, identifying protocols that specifically differentiate human ‘coronary-like’ VSMCs [9]. However, a more readily accessible source of VSMC progenitors is the skin-derived precursor (SKP), which is a progenitor cell residing in the dermal papilla and sheath of mouse and human hair follicles, in addition to hairless human skin such as foreskin [1012]. SKPs are Sox2+ progenitor cells that can be expanded as spheres in suspension, and can be directed to differentiate into neurons, Schwann cells, and adipocytes [10,11]. Importantly, we showed that in the presence of transforming growth factor-β (TGF-β)1 or TGF-β3, human foreskin-derived SKP can differentiate almost exclusively into functional VSMC in serum-free, normoglycemic conditions [13]. A major practical advantage of SKPs (as compared with skin-derived iPSCs) is that no reprogramming is required to generate VSMCs. However, as our earlier studies only used SKPs obtained from children [13], it was not known whether SKPs could be successfully isolated from older adults with CVD, or whether SKP-derived VSMC from such subjects might model their underlying CVD in vitro. The present study sought to address these questions, and to detail a simple systematic method to model dysfunctional VSMC physiology attributable to a cardiovascular risk factor such as T2D.

While progress has been made in modeling human disease with iPSC, most of these studies have been limited to elucidating the disease mechanisms of rare genetic disorders [1416]. Moreover, ectopic expression of the reprogramming factors (c-Myc, Klf4, Oct4, and Sox2) needed to induce iPSC from human somatic cells also causes significant epigenetic modifications [17,18]. Although iPSC do retain some epigenetic memory [19,20], the ‘embryonic’ nature of iPSC [21] is unlikely to model chronic, environmentally induced, and age-associated disorders such as T2D. Given these limitations, harvesting adult stem cell populations without reprograming from accessible tissue sources may better enable the modeling of such diseases.

Here, we report the ability to isolate SKPs from an older adult population to test the feasibility of attaining VSMC with disease signatures. Using live cell calcium imaging and impedance-based cellomics to demonstrate responsiveness to agonist treatment, as well as wound healing models in vitro, we demonstrate that SKP-derived VSMC from subjects with T2D retain cell physiological signatures that represent a unique platform for studying the molecular and cellular pathophysiology underlying the ‘metabolic memory’ of T2D.

Our data reveal that SKPs from older adults can be differentiated into VSMC at efficiencies similar to those of SKP derived from children [13]. However, subjects with T2D have fewer SKPs/g skin than subjects without T2D, suggesting that prolonged hyperglycemia and/or other metabolic abnormalities of T2D affect the abundance of dermal sphere-forming progenitors in adult skin. Despite this, SKPs from patients with T2D can be differentiated into VSMC at efficiencies similar to those of SKPs from patients without T2D. Despite prolonged normoglycemic culture conditions, SKP-derived VSMCs from patients with T2D demonstrated persistent alterations in calcium cycling, were more responsive to adrenergic agonists, and manifest impaired wound healing responses in vitro. Although SKP-derived VSMCs functioned distinctly from aortic- and saphenous vein-derived VSMCs as evidenced through calcium handling, SKP-derived VSMCs from subjects with T2D did resemble vessel-derived VSMCs from subjects with T2D. Specifically, defects in wound closure assays were observed in both SKP- and vessel-derived VSMCs from subjects with T2D. The present study reveals that SKP-derived VSMCs retain phenotypic signatures of T2D, and represent a promising platform for modeling the cellular pathophysiology of this disease.

Methods

Patients and samples

One hundred sixty-four patients undergoing cardiothoracic or thoracic surgery at Toronto General Hospital were recruited out of 172 that were approached for informed consent under an institutionally approved research protocol (UHN REB protocol 11-0841-TE). Complete clinical data and adequate skin biopsies were obtained from 158 patients, which formed our study population. Patients underwent surgery for diverse conditions including coronary artery disease (CAD), aortic stenosis, mitral regurgitation, aortic repair, lung cancer, and others (Table 1). Isolation of SKP from human skin was performed as previously described [12] and plated at 150,000–500,000 cells/ml in SKP proliferation medium (low glucose DMEM/Ham’s F12 3:1, 1% penicillin/streptomycin, 1 μg/ml Fungizone, 2% B27, 40 ng/ml FGF2, and 20 ng/ml EGF) [12]. Human SKPs were passaged as described [12] using collagenase XI for 30 min in a 37°C water bath and plated at 50,000 cells/ml into SKP proliferation medium supplemented with 50% filtered conditioned medium. Conditioned medium was added in order to promote optimal growth of SKPs by providing additional paracrine factors. In order to calculate SKP isolation efficiency, cell numbers were normalized to gram weight of skin.

Table 1
Patient demographics and co-morbidities in study population and by diagnosis of T2D
Patient factorsTotal (N=158) N (%)No T2D (N=100) N (%)T2D (N=58) N (%)P-value (T2D versus no T2D)Test
Age (years) 62.7 ± 13 62.6 ± 15 62.8 ± 8.3 0.91 
Male 114 (72.2) 70 (70.0) 44 (75.9) 0.77 
T2D 58 (36.7) No Yes N/A N/A 
HTN 110 (69.6) 63 (63.0) 47 (81.0) 0.01 
Dyslipidemia 101 (63.9) 56 (56.0) 45 (77.6) 0.003 
Smoker 33 (20.9) 20 (20.0) 13 (22.4) 0.70 
CAD 120 (75.9) 68 (68.0) 52 (89.7) 0.002 
MI 31 (19.6) 22 (22.0) 9 (15.5) 0.34 
Angina 17 (10.8) 10 (10.0) 7 (12.1) 0.67 
CHF 30 (19.0) 15 (15.0) 15 (25.9) 0.09 
Pulmonary HTN 7 (4.4) 2 (2.0) 5 (8.6) 0.05 
PAD 14 (8.9) 7 (7.0) 7 (12.1) 0.27 
Carotid stenosis 9 (5.7) 1 (1.0) 8 (13.8) 0.0008 
TIA or CVA 14 (8.9) 8 (8.0) 6 (10.3) 0.60 
Aortopathy 24 (15.2) 22 (22.0) 2 (3.4) 0.002 
CKD 7 (4.4) 2 (2.0) 5 (8.6) 0.05 
MVD 17 (10.8) 14(14.0) 3(5.2) 0.09 
AVD 33 (20.9) 26(26.0) 7(12.1) 0.04 
Congenital 11 (7.0) 9(9.0) 2(3.4) 0.19 
Myxoma 4 (2.5) 4(4.0) 0(0.0) 0.12 
Endocarditis 2 (1.3) 2 (2.0) 0 (0.0) 0.28 
AF 22 (13.9) 15 (15.0) 7 (12.1) 0.62 
Patient factorsTotal (N=158) N (%)No T2D (N=100) N (%)T2D (N=58) N (%)P-value (T2D versus no T2D)Test
Age (years) 62.7 ± 13 62.6 ± 15 62.8 ± 8.3 0.91 
Male 114 (72.2) 70 (70.0) 44 (75.9) 0.77 
T2D 58 (36.7) No Yes N/A N/A 
HTN 110 (69.6) 63 (63.0) 47 (81.0) 0.01 
Dyslipidemia 101 (63.9) 56 (56.0) 45 (77.6) 0.003 
Smoker 33 (20.9) 20 (20.0) 13 (22.4) 0.70 
CAD 120 (75.9) 68 (68.0) 52 (89.7) 0.002 
MI 31 (19.6) 22 (22.0) 9 (15.5) 0.34 
Angina 17 (10.8) 10 (10.0) 7 (12.1) 0.67 
CHF 30 (19.0) 15 (15.0) 15 (25.9) 0.09 
Pulmonary HTN 7 (4.4) 2 (2.0) 5 (8.6) 0.05 
PAD 14 (8.9) 7 (7.0) 7 (12.1) 0.27 
Carotid stenosis 9 (5.7) 1 (1.0) 8 (13.8) 0.0008 
TIA or CVA 14 (8.9) 8 (8.0) 6 (10.3) 0.60 
Aortopathy 24 (15.2) 22 (22.0) 2 (3.4) 0.002 
CKD 7 (4.4) 2 (2.0) 5 (8.6) 0.05 
MVD 17 (10.8) 14(14.0) 3(5.2) 0.09 
AVD 33 (20.9) 26(26.0) 7(12.1) 0.04 
Congenital 11 (7.0) 9(9.0) 2(3.4) 0.19 
Myxoma 4 (2.5) 4(4.0) 0(0.0) 0.12 
Endocarditis 2 (1.3) 2 (2.0) 0 (0.0) 0.28 
AF 22 (13.9) 15 (15.0) 7 (12.1) 0.62 

AF, atrial fibrillation; AVD, aortic valve disease; C, Chi-squared test; CHF, congestive heart failure; CKD, chronic kidney disease; MVD, mitral valve disease; PAD, peripheral arterial disease; T, t-test; TIA or CVA, transient ischemic attack or cerebrovascular attack. Bold text indicates statistical significance at P≤0.05.

Isolation of vessel-derived VSMCs

Aortic VSMCs were obtained from subjects undergoing coronary artery bypass grafting (CABG) (buttons) and thoracic aortic repair. Saphenous vein was obtained from subjects undergoing CABG. VSMCs from the aorta were peeled off of the vessel and finely minced with scissors. Saphenous vein was minced whole and aortic buttons were not minced due to their small size. Tissue was incubated in 5 ml of 0.1 mg/ml collagenase II (Worthington) for 10 min in a 37°C water bath. Tissue was passed several times through a 10 ml pipette to dislodge cells. A further 5 ml of collagenase II was added, and the sample was incubated in a 37°C water bath for another 10 min. After trituration, 10 ml of collagenase II was added for a final 10 min incubation in the 37°C water bath. The cell suspension was passed through a 40 μm cell strainer (BD Biosciences) and the cells centrifuged at 300 g for 7 min. The aortic buttons were not passed through a cell strainer but centrifuged at 300 g for 7 min. After the supernatant was discarded, the cell pellet was added to a T25 with 10 ml of M231 medium (Thermo Fisher) supplemented with Smooth Muscle Growth Supplement (Thermo Fisher). Aortic buttons were added to a T12.5 flask with 10 ml of M231 medium supplemented with Smooth Muscle Growth Supplement.

Differentiation

All differentiated VSMCs were derived from SKPs at passage 2 (P2). SKPs were differentiated as described in 1% FBS with 2 ng/ml TGF-β1 or -β3 in low glucose DMEM/Ham’s F12 3:1 with 1% penicillin/streptomycin [13].

Indirect immunofluorescence

SKP-derived VSMCs were identified as previously detailed [13]. Cells were incubated overnight at 4°C with primary antibodies against α-smooth muscle actin (ASMA) (1:800, A2547 clone 1A4 Sigma), Calponin (1:250, Abcam), and Smoothelin B (1:250, Santa Cruz). Cells were imaged with an Olympus FV1000 laser scanning confocal microscope.

Real-time cell analysis

Real-time changes in electrical impedance were monitored using an xCELLigence system (Acea Biosciences). Cells were seeded in duplicate onto 16-well E-plates at 5,000 cells per well and differentiated for 7–12 days in 1% FBS with 2 ng/ml TGF-β3. After differentiation, cells were treated with concentrations of phenylephrine (PE) or norepinephrine (NE) ranging from 10−11 to 10−1 mmol/l with changes in impedance monitored every 15 s. Using the xCELLigence RTCA software (version 2.0), sigmoidal curves were generated. EC50 values were calculated from the SKP-derived VSMCs of individual patients.

Calcium imaging

Calcium imaging of SKP-derived VSMCs was performed as previously described [9]. Cells were incubated with 5 μM of the membrane-permeable Ca2+-sensitive dye Fluo-5F AM (Life Technologies) and 0.1% (w/v) Pluronic F-127 (Life Technologies) for 45 min at 37°C and 5% CO2 in a humidified incubator. Cells were subsequently washed three times for 5 min with Hank’s Balanced Salt Solution (HBSS). All Ca2+ imaging were performed using HBSS as a cell culture replacement in an environmental chamber at 37°C and 5% CO2 using an Olympus FV1000 confocal microscope. Images were recorded continually until the experiment was completed. Laser excitation was at 488 nm and emission was recorded at 516 nm. Baseline (2 min) was recorded for each sample, before the addition of PE (10 μM) for 10 min of recording. Following this, NE (10 μM) was added to untreated cells in adjacent wells for an additional 10 min of recording. Video files were analyzed for fluorescence intensity using Fluoview1000 software. Frequency, baseline intensities, and peak area under the curve were calculated using GraphPad (Prism) version 5.04. Between 4 and 25 cells were analyzed per human subject per experimental condition. Data shown are means of cellular responses from individual patients.

Wound healing

SKPs were differentiated as above and plated on four-well chamber slides at 20,000 cells/cm2 and cultured for 10–14 days to achieve a confluent culture. A scratch wound was then generated by a pipette tip, and the culture was imaged on a Zeiss ApoTome microscope every 10–30 min for 2–5 days in an environmental chamber at 37°C and 5% CO2. To calculate the time of wound closure, the area (pixels2) unoccupied by cells for each time point was calculated using Image Pro Premiere (version 9.1).

Statistics

Continuous variables are presented as means with standard deviations (SD), while categorical variables are presented as frequencies or proportions as appropriate. Univariate analyses were performed to assess the association between PE EC50, NE EC50, and wound healing data. Normally distributed data were analyzed using Student’s t-test with Mann–Whitney test was used for data without normal distribution. Multivariable analyses were performed using linear regression to determine the association between co-morbidities and absolute numbers of cells at passage 0, P1, and P2 (Tables 2 and 3) as well as the proportion of cells at P0, P1, and P2 (Table 4). Independent variables were chosen based on clinical relevance, and variables found to be strongly associated with outcomes in univariate analyses. These were: age, sex, T2D, HTN, dyslipidemia, CAD, PAD, pulmonary HTN, history of cigarette smoking, and site of tissue extraction. Due to the non-normality of residuals, we modeled the relationship between the baseline variables and the logarithm of the outcome variables specified. Linear regression analysis was conducted using SAS Version 9.4 (SAS Institute, Cary, NC). P<0.05 was considered statistically significant (Tables 24).

Table 2
Multivariate analysis of predictors of initial skin cell yield
PredictorP-value
Male 0.657 
Age 0.157 
T2D 0.836 
HTN 0.331 
Dyslipidemia 0.130 
Smoker 0.049 
CAD 0.038 
Pulmonary HTN 0.567 
PAD 0.933 
PredictorP-value
Male 0.657 
Age 0.157 
T2D 0.836 
HTN 0.331 
Dyslipidemia 0.130 
Smoker 0.049 
CAD 0.038 
Pulmonary HTN 0.567 
PAD 0.933 

Bold text indicates statistical significance at P≤0.05.

Results

Of 172 elective surgery patients approached with an institutionally approved research protocol, 164 provided informed consent and were enrolled in the study (Figure 1). Of these, complete clinical and SKP abundance data were obtained from 158 subjects, with a mean age of 62.7 ± 13 years, and the majority being male (72%) (Table 1). All enrolled patients underwent cardiac or thoracic surgery for indications as varied as aortic aneurysm repair, valve replacement, CABG, and lung nodule resection. The majority of patients had HTN (70%) and dyslipidemia (64%), with 37% having T2D (Table 1). Consistent with numerous epidemiological studies, the prevalence of HTN, dyslipidemia, CAD, carotid stenoses, chronic kidney failure, and AVD was greater in subjects with T2D (Table 1) [22,23]. In agreement with one previous report [24], we also found the prevalence of pulmonary HTN to be greater in subjects with T2D (Table 1). There was also an inverse correlation between T2D and aortic aneurysm consistent with another report [25].

Study overview

Figure 1
Study overview

The number of subjects recruited following informed consent in whom complete clinical information and an adequate skin biopsy was obtained is shown. These subjects were stratified into those with known T2D and those without T2D (No T2D). The number of skin samples obtained from leg and chest incisions is also provided. The yield of SKPs was lower in subjects with T2D at both first (P1) and second passages (P2). *P<0.01, **P<0.05 T2D versus No T2D by Chi-Square.

Figure 1
Study overview

The number of subjects recruited following informed consent in whom complete clinical information and an adequate skin biopsy was obtained is shown. These subjects were stratified into those with known T2D and those without T2D (No T2D). The number of skin samples obtained from leg and chest incisions is also provided. The yield of SKPs was lower in subjects with T2D at both first (P1) and second passages (P2). *P<0.01, **P<0.05 T2D versus No T2D by Chi-Square.

To determine whether SKPs could be isolated from this older adult cohort, a <1 cm2 incisional skin biopsy was weighed (mean ± SD: 0.584 ± 0.384 g) and digested with a commercial enzyme cocktail (Blendzyme™, Roche). First, we quantified the number of cells in the skin biopsy. As shown in Figure 2A, the number of cells that can be harvested from a skin biopsy was not dependent on age (R2 = 0.0024, P=0.11). We next performed multivariate linear regression of co-morbidities with skin cell harvest. This analysis demonstrated that smoking and CAD had an independent negative effect on the number of cells that could be isolated from skin biopsies (Table 2). Importantly, T2D was not associated with a difference in the total number of cells that could be isolated from skin.

Subjects with T2D have fewer SKPs/g skin

Figure 2
Subjects with T2D have fewer SKPs/g skin

(A) The number of cells initially harvested from patient skin biopsies was counted for each subject and normalized to skin weight (g). Advancing age does not have a significant effect on the number of cells initially harvested from skin biopsies from subjects with and without T2D. (B) Subjects with T2D have fewer SKPs/g skin. The number of SKPs isolated after passage 1 (P1) were quantified and normalized to skin weight (g). Adult subjects with T2D have significantly fewer SKPs/g skin than subjects without T2D (*P<0.05).

Figure 2
Subjects with T2D have fewer SKPs/g skin

(A) The number of cells initially harvested from patient skin biopsies was counted for each subject and normalized to skin weight (g). Advancing age does not have a significant effect on the number of cells initially harvested from skin biopsies from subjects with and without T2D. (B) Subjects with T2D have fewer SKPs/g skin. The number of SKPs isolated after passage 1 (P1) were quantified and normalized to skin weight (g). Adult subjects with T2D have significantly fewer SKPs/g skin than subjects without T2D (*P<0.05).

Subsequent to this, we assayed the number of SKPs (i.e. spheres emerging in our defined SKP media [13]) that could be isolated from the initial skin cell harvest. SKPs could be harvested from 64% of all patients at P1. This number declined to 39% at P2 (Figure 1). Not only did the percentage of subjects yielding SKPs drop with each passage, so did the total number of SKPs. When comparing the mean number of SKPs (normalized to g weight of skin) cultured at P1 versus P2 from the total cohort, we observed an overall 25% decline in the number of SKPs available at P2. In an attempt to identify clinical factors that predict overall SKP yield, we compared skin weight-normalized SKP numbers by co-morbidity using multivariate regression. In this analysis, factors such as gender or conditions such as dyslipidemia, smoking, CAD, pulmonary-HTN, or peripheral arterial disease (PAD) did not predict SKP yield, while T2D and systemic-HTN did (Table 3).

Table 3
Multivariate analysis of predictors of overall SKP yield
PredictorP-value
Male 0.540 
T2D <0.001 
HTN 0.045 
Dyslipidemia 0.371 
Smoker 0.107 
CAD 0.438 
Pulmonary HTN 0.655 
PAD 0.140 
PredictorP-value
Male 0.540 
T2D <0.001 
HTN 0.045 
Dyslipidemia 0.371 
Smoker 0.107 
CAD 0.438 
Pulmonary HTN 0.655 
PAD 0.140 

Bold text indicates statistical significance at P≤0.05.

When we stratified our cohort into subjects with or without T2D, we found that we could culture SKPs from 72% of adult subjects without T2D at P1, but only 47% of these patients at P2 (Figure 1). For subjects with T2D, only 48% of the subjects yielded SKPs at P1 and this number declined to 24% at P2 (Figure 1). Thus, patients with T2D account for the majority of failed SKP isolations, with a Chi-squared analysis of the decrease in SKP isolation frequency (between P1 and P2) revealing a significant effect of T2D (P<0.05 and P<0.01). Of interest, this effect of T2D on SKP yield was independent of age (T2D: 62.8 ± 8.3 years versus no T2D: 62.6 ± 15 years, P=0.910), just as there was no effect of increasing age on skin cell number, age did not differ in subjects with and without T2D (Figure 2A). Numerically, in adults without T2D, 1.0% of the total skin cells isolated formed SKPs at P1 (N=100), compared with only 0.49% in subjects with T2D (N=58, P<0.05) (Figure 2B).

We next assessed whether any other co-morbidities affected the ability of skin cells to survive or grow into SKPs, by multivariate regression on the ratio of cells at P0 to the number of SKPs at P1. Again, only T2D emerged as a significant factor affecting the ability of skin cells to generate SKPs (Table 4). When the number of cells at P2 was subtracted from the number of cells at P1, T2D also emerged as the only significant factor affecting the ability of SKPs to passage (Table 4).

Table 4
Multivariate analysis of predictors of SKP yield after passage from P0 to P1 and P1 to P2
PredictorP-value (P0 to P1)P-value (P1 to P2)
Male 0.548 0.411 
Age 0.215 0.485 
T2D 0.026 0.0009 
HTN 0.144 0.939 
Dyslipidemia 0.591 0.478 
Smoker 0.512 0.154 
CAD 0.151 0.975 
Pulmonary HTN 0.718 0.890 
PAD 0.109 0.474 
PredictorP-value (P0 to P1)P-value (P1 to P2)
Male 0.548 0.411 
Age 0.215 0.485 
T2D 0.026 0.0009 
HTN 0.144 0.939 
Dyslipidemia 0.591 0.478 
Smoker 0.512 0.154 
CAD 0.151 0.975 
Pulmonary HTN 0.718 0.890 
PAD 0.109 0.474 

Bold text indicates statistical significance at P≤0.05.

We then wanted to determine whether SKPs from older adults could differentiate into VSMC at the high efficiencies and serum-free conditions previously observed in foreskin-derived SKPs from children [13]. SKPs were differentiated with TGF-β3, with or without 1% serum for 7–12 days. In the presence of 1% serum and TGF-β3, SKPs differentiated almost exclusively into VSMC as evidenced by double-staining with either ASMA+ and Calponin+ or ASMA+ and Smoothelin-B (SmthnB+) (Figure 3A). As we are not able to control for fluorescence intensity, observed differences in staining intensity between these markers (e.g. calponin and SmthnB) are of uncertain significance. Nevertheless, based on double staining, differentiation efficiencies in the presence of 1% serum and TGF-β3 reached 85–98% as compared with 13–17% in the presence of 1% serum alone (Figure 3A). Of interest, in contrast with our report on foreskin-derived SKPs from children [13], SKPs from this older adult population failed to survive in serum-free conditions, even in the presence of TGF-β1 or -β3. Despite differences in the abundance of SKPs in the skin between subjects with and without T2D, their ability to differentiate almost exclusively into VSMC did not differ (Figure 3B). Based on ASMA+ Calponin+ or ASMA+ SmthnB+ double staining, SKPs from subjects with T2D also differentiated into VSMC at 89–98% efficiency (Figure 3B). As stated above, it is difficult to comment on the biological significance of qualitative differences in the staining intensities of specific SMC-markers in SKP-derived VSMC from subjects with and without T2D (Figure 3B versus 3A).

SKPs from adults with and without T2D can be differentiated into VSMCs at high efficiency

Figure 3
SKPs from adults with and without T2D can be differentiated into VSMCs at high efficiency

SKPs from subjects with (A) and without (B) T2D were differentiated for 7–12 days. Differentiated SKPs were stained for ASMA, Calponin, and Smthn B. SKP-derived VSMC from adult subjects with and without T2D differentiated to >80% efficiencies in 1% FBS supplemented with TGF-β3. Representative photomicrographs are shown; scale bar = 60 μm. The number of cells staining double-positive for VSMC markers (AMSA+ Calponin+ or ASMA+ Smthn B+) was quantified (N=4 subjects in each analysis). (C) Using an impedance-based real-time cell analyzer, the doubling times of SKP-derived VSMC from subjects with (N=7) or without (N=14) T2D did not differ (P = NS).

Figure 3
SKPs from adults with and without T2D can be differentiated into VSMCs at high efficiency

SKPs from subjects with (A) and without (B) T2D were differentiated for 7–12 days. Differentiated SKPs were stained for ASMA, Calponin, and Smthn B. SKP-derived VSMC from adult subjects with and without T2D differentiated to >80% efficiencies in 1% FBS supplemented with TGF-β3. Representative photomicrographs are shown; scale bar = 60 μm. The number of cells staining double-positive for VSMC markers (AMSA+ Calponin+ or ASMA+ Smthn B+) was quantified (N=4 subjects in each analysis). (C) Using an impedance-based real-time cell analyzer, the doubling times of SKP-derived VSMC from subjects with (N=7) or without (N=14) T2D did not differ (P = NS).

Using a cell impedance-based cellomics technology, which measures changes in cell adhesion and cell shape in real time as indicators of metrics such as proliferation [26], we noted no differences in proliferation of SKP-derived VSMCs from subjects with or without T2D over 7 days (Figure 3C). Specifically, a doubling time of 32 ± 3 h was observed in subjects without T2D versus a doubling time of 34 ± 5 h in subjects with T2D (Figure 3C; P = NS).

Since SKP-derived VSMCs obtained from subjects with or without T2D were morphologically identical despite SKPs being fewer in subjects with T2D, we sought to functionally compare SKP-derived VSMCs with vessed-derived VSMCs isolated from aorta or saphenous vein. SKP-derived VSMCs, in addition to aortic- and saphenous vein-derived VSMCs, were treated with Fluo-5F, a Ca2+-sensitive fluorophore, to observe Ca2+ flux in response to NE (10 µM). Changes in intracellular calcium concentrations are a recognized integrative physiology marker of many homeostatic cell functions, including pharmacomechanical coupling. Calcium imaging may also be used to distinguish VSMC from phenotypically similar but non-contractile fibroblasts and myofibroblasts. Calcium imaging was performed on a limited number of cell lines from individual patients. Taken together, our results demonstrate that SKP-derived VSMCs were functionally distinct from aortic- or saphenous vein-derived VSMCs. Significantly more SKP-derived VSMCs responded to NE treatment than aortic- or saphenous vein-derived VSMCs (31.4 ± 4.7%, N=19 versus 14 ± 2.5%, N=15, versus 14.5 ± 2.8%, N=14, P<0.01) (Figure 4A), and baseline fluorescence intensity was significantly higher in SKP-derived VSMCs than that of the aorta or saphenous vein (1619 ± 141, N=19 versus 508 ± 57, N=15 versus 485 ± 91, N=12, P<0.001) (Figure 4B). Moreover, the frequency of calcium waves (0.018 ± 0.003, N=27 versus 0.004 ± 0.0009, N=15 versus 0.003 ± 0.0004, N=12) and mean peak area (121,155 ± 14,670, N=27 versus 17,204 ± 2,929, N=11 versus 18,821 ± 4,931, N=11) of those calcium waves were significantly higher for SKP-derived VSMCs compared with aortic- or saphenous vein-derived VSMCs (P<0.0001) (Figure 4C and D). Similar results were observed for PE-treated cells (data not shown).

SKP-derived VSMCs are functionally distinct from aorta- and saphenous vein-derived primary VSMCs

Figure 4
SKP-derived VSMCs are functionally distinct from aorta- and saphenous vein-derived primary VSMCs

Cells were loaded with the calcium-sensitive dye Fluo-5F, treated with NE (10 μM), and visualized with a confocal microscope for 10 min to observe calcium flux. Significantly, more SKP-derived VSMCs responded to NE (A) than aorta- or saphenous vein-derived VSMCs. Baseline fluorescence intensity was significantly greater in SKP-derived VSMCs than aorta- or saphenous vein-derived VSMCs (B). SKP-derived VSMCs responded with greater frequency of calcium waves than aorta- or saphenous vein-derived VSMCs (C), and the mean peak area was significantly greater in SKP-derived VSMCs than aorta- and saphenous vein-derived VSMCs (D); **P<0.01, ***P<0.001, and ****P<0.0001. The number of subjects analyzed per group is indicated below the x-axis.

Figure 4
SKP-derived VSMCs are functionally distinct from aorta- and saphenous vein-derived primary VSMCs

Cells were loaded with the calcium-sensitive dye Fluo-5F, treated with NE (10 μM), and visualized with a confocal microscope for 10 min to observe calcium flux. Significantly, more SKP-derived VSMCs responded to NE (A) than aorta- or saphenous vein-derived VSMCs. Baseline fluorescence intensity was significantly greater in SKP-derived VSMCs than aorta- or saphenous vein-derived VSMCs (B). SKP-derived VSMCs responded with greater frequency of calcium waves than aorta- or saphenous vein-derived VSMCs (C), and the mean peak area was significantly greater in SKP-derived VSMCs than aorta- and saphenous vein-derived VSMCs (D); **P<0.01, ***P<0.001, and ****P<0.0001. The number of subjects analyzed per group is indicated below the x-axis.

We next sought to examine if SKP-derived VSMC from adults with T2D were functionally distinct. Thus, we divided our results into subjects with or without a diagnosis of T2D. Although SKP-derived VSMC from adults with T2D responded to NE with maximum Ca2+ amplitudes that did not differ from SKP-derived VSMC from subjects without T2D (data not shown), their baseline fluorescence levels (i.e. resting Ca2+ concentrations) were higher (1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05; Figure 5A) and their mean peak fluorescence area (i.e. total Ca2+ release) following NE trended to be greater (177,207 ± 24,669, N=7 versus 101,537 ± 15,881, N=20, P<0.08; Figure 5B), despite fewer Ca2+-dependent oscillations (events ms−1 cells−1: 0.011 ± 0.004, N=8 versus 0.021 ± 0.003, N=19, P<0.05; Figure 5C). Near identical differences were observed between SKP-derived VSMC from subjects with and without T2D in response to PE (10 μM) (data not shown).

SKP-derived as well as primary aorta- and saphenous vein-derived VSMCs from subjects with T2D have enhanced calcium responses

Figure 5
SKP-derived as well as primary aorta- and saphenous vein-derived VSMCs from subjects with T2D have enhanced calcium responses

(A) Baseline fluorescence intensity was greater in SKP-derived VSMCs isolated from subjects with T2D. (B) In response to NE (10 μM), the frequency of calcium waves (events/second per cell) was lower in SKP-derived VSMCs isolated from subjects with T2D. (C) However, mean peak area of the calcium response (sec × fluorescence intensity × 1000) trended higher in SKP-derived VSMCs from subjects with T2D (P<0.08). Aorta- and saphenous vein-derived VSMCs also manifest increased mean peak area of their calcium response to NE (10 μM) in subjects with T2D (D and E); *P<0.05. The number of subjects analyzed per group is indicated below the x-axis.

Figure 5
SKP-derived as well as primary aorta- and saphenous vein-derived VSMCs from subjects with T2D have enhanced calcium responses

(A) Baseline fluorescence intensity was greater in SKP-derived VSMCs isolated from subjects with T2D. (B) In response to NE (10 μM), the frequency of calcium waves (events/second per cell) was lower in SKP-derived VSMCs isolated from subjects with T2D. (C) However, mean peak area of the calcium response (sec × fluorescence intensity × 1000) trended higher in SKP-derived VSMCs from subjects with T2D (P<0.08). Aorta- and saphenous vein-derived VSMCs also manifest increased mean peak area of their calcium response to NE (10 μM) in subjects with T2D (D and E); *P<0.05. The number of subjects analyzed per group is indicated below the x-axis.

When aortic and saphenous vein-derived VSMCs were stratified according to T2D, it was found that they resembled SKP-derived VSMCs in that subjects with T2D demonstrated significantly greater mean peak area (sec × fluorescence intensity; aortic VSMCs: 10,570 ± 2,911, N=6 versus 22,695 ± 5,191, N=5; P<0.05; saphenous vein VSMCs: 6,719 ± 1,032, N=4 versus 18,091 ± 3,928, N=6; P<0.01) (Figure 5D and E). Together, these data suggest that SKP-derived VSMCs from subjects with T2D have enhanced sensitivity to adrenergic agonists similar to aortic- and saphenous vein-derived VSMCs. Again, the groups tested in this analysis (i.e. T2D versus No T2D) did not differ significantly with respect to age, sex, or other co-morbidities.

To confirm the increased sensitivity to adrenergic agonists of SKP-derived VSMCs with T2D, we performed a parallel assay using our impedance-based cellomics approach. Whereas monitoring changes in cell impedance over days typically reflects changes in cell proliferation, monitoring changes of this parameter over seconds can detect changes in cell shape and adhesion. Here, SKP-derived VSMCs were treated with increasing concentrations of PE or NE (100 to 10−8 μM) to obtain EC50 values of real-time dose-dependent changes in electrical impedance over seconds. Interestingly, SKP-derived VSMC from subjects with T2D demonstrated enhanced responsiveness to PE and NE treatment as manifested by a lower EC50 value for each agent (PE: 72.3 ± 63.6 nM, N=5 versus 3,684 ± 3,122 nM, N=9, P<0.05; NE: 11.71 ± 11 nM, N=5 versus 413.6 ± 131 nM, N=9, P<0.005) (Figure 6A and B). Although relatively small in number, the subjects chosen for this analysis resemble the overall patient population and did not appreciably differ between groups (No T2D: age 62 ± 8 years, sex M:F 8:1, surgery: CABG:Valve 8:1; T2D: age 63 ± 14 years, sex M:F 4:1, surgery: CABG:Valve 3:2) suggesting that alterations in their responsiveness to vasoconstrictors, even with prolonged normoglycemic culture conditions, were attributable to their underlying T2D rather than other clinical factors.

SKP-derived VSMCs from subjects with T2D have altered sensitivity to adrenergic agonists and impaired wound healing

Figure 6
SKP-derived VSMCs from subjects with T2D have altered sensitivity to adrenergic agonists and impaired wound healing

(A) SKP-derived VSMCs from subjects with and without T2D were treated with increasing concentrations of PE (A) or NE (B) to generate EC50 values of changes in cell size and shape as measured by real-time electrical impedance (measured every 15 s and averaged over 15 min). SKP-derived VSMCs from subjects with T2D have enhanced sensitivity to PE (*P<0.05) and NE (**P<0.005). (C) Wound closure was assessed in SKP-derived VSMCs from subjects with and without T2D in a wound healing chamber. Percent area wound closure at 24 h is plotted (*P<0.05). A total of four wounds per subject were examined. SKP-derived VSMCs from subjects with T2D have impaired wound closure. The number of subjects per group is indicated below the x-axis. Similar trends in delayed wound closure were also observed in aorta- and saphenous vein-derived VSMCs (see manuscript text for details). Magnification bar = 30 μm.

Figure 6
SKP-derived VSMCs from subjects with T2D have altered sensitivity to adrenergic agonists and impaired wound healing

(A) SKP-derived VSMCs from subjects with and without T2D were treated with increasing concentrations of PE (A) or NE (B) to generate EC50 values of changes in cell size and shape as measured by real-time electrical impedance (measured every 15 s and averaged over 15 min). SKP-derived VSMCs from subjects with T2D have enhanced sensitivity to PE (*P<0.05) and NE (**P<0.005). (C) Wound closure was assessed in SKP-derived VSMCs from subjects with and without T2D in a wound healing chamber. Percent area wound closure at 24 h is plotted (*P<0.05). A total of four wounds per subject were examined. SKP-derived VSMCs from subjects with T2D have impaired wound closure. The number of subjects per group is indicated below the x-axis. Similar trends in delayed wound closure were also observed in aorta- and saphenous vein-derived VSMCs (see manuscript text for details). Magnification bar = 30 μm.

We next assessed the ability of SKP-derived VSMC from adults with or without T2D to heal scratch wounds made to confluent cultures in vitro. In this assay, SKP-derived VSMC from subjects with T2D had reduced ability to close a wound 24 h after injury, as compared with SKP-derived VSMCs from adults without T2D, which closed their wounds more completely during that time period (% closure: 67.04 ± 10.3, N=4 versus 21.89 ± 3.6, N=4, P<0.05) (Figure 6C). Reduced % wound closure was also observed in vessel-derived primary VSMCs 24 h after injury, although this did not achieve statistically significance (aorta: 68.3 ± 7.0%, N=8 versus 45.7 ± 11.4%, N=4, P=0.125; saphenous vein: 54.7 ± 10.0%, N=5 versus 39.4 ± 19.1%, N=5, P=0.250) (data not shown). In this analysis, although the subjects with T2D were somewhat younger (age: 51 ± 5 years, sex: M:F, 3:1, surgery: CABG:Valve 4:0), their SKP-derived VSMC showed less effective wound closure than SKP-derived VSMC from older subjects without T2D (No T2D: age: 63 ± 14 years, sex: M:F 3:1, surgery: CABG:Valve 4:0).

Discussion

In the present study, we were able to isolate dermal progenitor cells (SKPs) from the skin of adults with a variety of cardiovascular and thoracic conditions with and without T2D. Although T2D decreased the abundance of human SKPs, the latter could still be differentiated into VSMCs at efficiencies similar to subjects without T2D. However, VSMC from subjects with T2D displayed key functional differences as compared with those without T2D. Similar to a study in which uterine artery-derived primary VSMCs were freshly isolated from adults with T1D [27], differentiated VSMC from SKPs of adults with T2D continued to display altered calcium handling even after weeks of tissue culture in normoglycemic conditions. Moreover, despite prolonged culture ex vivo, SKP-derived VSMC from subjects with T2D retained enhanced sensitivity to PE and NE. Finally, SKP-derived VSMC from adults with T2D displayed impaired wound healing responses compared with those without T2D. These persistent (i.e. prolonged tissue-culture resistant) findings suggest that skin-localized progenitor cells harbor molecular genetic signatures of disease and/or age that can be transmitted to subsequent passages of differentiated VSMCs—even after prolonged normoglycemic culture. When compared with primary VSMCs isolated from aorta or saphenous vein, SKP-derived VSMCs functioned distinctly with regards to their calcium handling properties. However, like aorta- and saphenous vein-derived VSMCs, SKP-derived VSMCs exhibited a phenotype closely associated with T2D.

SKPs have been shown to be involved in dermal homeostasis and wound healing [28,29]. It remains unanswered whether the known impaired wound healing responses in patients with T2D are due to the present study’s demonstration of (a) reduced numbers of SKPs present in skin or (b) functional abnormalities of the VSMCs derived from SKPs.

Exposure of the vasculature to hyperglycemia and other metabolic disturbances is believed to leave a persistent imprint on vascular cells including the VSMC. This premise underlies their dysfunction and potentially also the macrovascular complications so commonly described among subjects with T2D. Indeed, VSMCs play important roles in the pathophysiology of the atherosclerosis that underlies CAD, cerebrovascular disease, and PAD [30]. In HTN as well, VSMCs participate in the pathophysiology of elevated systemic vascular resistance and blood pressure by manifesting enhanced contractile properties and sensitivity to sympathomimetic stimulation [31]. HTN, in turn, contributes to injury of the vascular endothelium by increasing the permeability of the vessel wall to lipoproteins that accelerate atherosclerosis [32], and the production of extracellular matrix by VSMC [33]. VSMCs also secrete pro-inflammatory cytokines and superoxide that increase the recruitment of inflammatory cells and promote an environment that further accelerates atherosclerosis and the risk of plaque rupture [34]. Overall, VSMCs play a large role in the pathophysiology of atherosclerosis and macrovascular complications. Yet current animal models fail to adequately recapitulate these processes, and better human model systems are needed.

T2D and its associated metabolic abnormalities have been shown to have deleterious effects on VSMCs. In human arterial SMCs derived from subjects with T2D, increased proliferation was observed due to their increased secretion of mitogenic factors [35,36]. Such VSMC also showed accelerated cell cycle entry, which was accompanied by post-translational modifications to specific intracellular signaling pathways [37]. However, in saphenous vein-derived VSMCs from subjects with T2D, the opposite effect was observed with lower VSMC proliferation rates [3840]. Interestingly, no significant differences in phenotype or function were observed in VSMC derived from the internal mammary artery of subjects with or without T2D [40]. In our study, we found no differences in proliferation rates between VSMCs derived from the SKPs of subjects with or without T2D. However, these same SKP-derived VSMC showed significant functional differences in parameters such as intracellular calcium handling, PE and NE sensitivity, and wound healing. This highlights the functional heterogeneity of VSMCs derived from venous versus arterial sources, as well as different anatomical locations, and now SKPs. It is interesting to note that SKP-derived VSMCs displayed more robust calcium handling and wound healing abnormalities than vessel-derived VSMCs, suggesting that skin may be a tissue that retains ‘more’ metabolic memory than the vasculature itself.

It is especially intriguing that despite prolonged culture in normoglycemic conditions (∼5 mM glucose) SKP-derived VSMC from adults with T2D retained cell physiological differences even weeks after being removed from the patient. Indeed, our in vitro demonstration of such a ‘metabolic memory’ or ‘legacy effect’ may be exploited to determine why subjects with T2D continue to experience high rates of macrovascular complications despite improvements in glycemic controls [41]. Whether this legacy effect is attributable to epigenetic or structural changes to the vasculature remains unclear, but it is a question that SKP-derived VSMC may help to answer.

Our results provide the first evidence that T2D affects the abundance of dermal stem cells in human skin, a finding that may underlie the mechanism by which subjects with T2D experience impaired wound healing. Moreover, the impaired wound healing that T2D VSMCs exhibit in vitro is consistent with the clinical situation where subjects with T2D are known to have impaired wound healing [42,43]. These findings also provide justification for further studies that restore or stimulate SKPs for the treatment of chronic wounds. In addition, our study is the first to demonstrate that an adult progenitor cell such as the SKP can be used to generate VSMC that model phenotypes induced by T2D. Here, we report a method of studying the vasculature in older subjects with T2D that is minimally invasive. In the absence of osmotic or hyperglycemic conditions, these cells maintain an enduring T2D-associated phenotype that resists differentiation and length of culture.

Our analyses also suggest an independent effect of HTN on SKP yield. As such, we may also have a system for modeling human VSMC physiological abnormalities underlying HTN in vitro, although further studies are needed to better define the differences that exist in SKP-derived VSMC of subjects with versus without-HTN.

There are several limitations to the present study. First, we are not able to cryopreserve SKP-derived VSMC. As a result, our physiological experiments were limited in duration (∼6 weeks), and experiments had to be performed in a relatively compressed time period. Also, although SKP isolation efficiencies were robustly determined, we could not perform additional serial passaging of SKPs due to diminishing cell yield. Vessel-derived VSMCs were also not cultured in the same medium as SKP-derived VSMCs that may have an effect on the calcium handling properties of these cells. Moreover, at this stage of our studies, we do not yet have a molecular mechanism for why SKP-derived VSMC from subjects with T2D exhibit enhanced sensitivity to adrenergic agonists, and we do not know why or how the cells exhibit ‘metabolic memory’. Further analysis of the expression of adrenergic receptors, as well as components of the calcium signaling pathway in SKP-derived VSMCs may define molecular perturbations underlying the phenotypes documented in this report. We hypothesize that the metabolic memory of SKP-derived VSMCs may depend on specific epigenetic modifications. Future studies in this model, and parallel animal model systems, are needed to unravel this observation, and potentially inform which signaling pathways in progenitors and/or VSMCs are modified by T2D. Importantly, we were only able to perform functional (VSMC) analyses on a limited number of study subjects (Figure 1). This was primarily related to whether subject skin samples yielded sufficient numbers of SKPs for subsequent analysis. As such, we recognize that our analyses may be biased toward those patients with milder defects in SKP abundance.

Finally, it is tempting to speculate that the relatively simple approach identified in this report may prove useful for drug screening, earlier diagnosis of T2D-related vascular disorders, and perhaps even the identification of patients at greatest risk of cardiovascular events. Our strategy may also enable ‘pharmaco/physiomics’ capable of identifying more favorable treatment options for individual subjects, and herald more individualized approaches to cardiovascular medicine (particularly in subjects with T2D), which currently lag behind individualized approaches to cancer and other conditions.

Clinical perspectives

  • We previously demonstrated directed differentiation of skin-derived precursors (SKPs) from children into vascular smooth muscle cells (VSMCs). Here we define that SKPs can also be obtained from older subjects with or without type-2 diabetes (T2D), and while their differentiation capacity into VSMC was not altered by this metabolic condition, they retain signatures of this disease in vitro.

  • We also demonstrated that SKPs are less abundant in subjects with T2D, providing a clinical basis for the impaired wound healing observed in this population. Although differentiation capacity of SKPs from subjects with T2D was not altered, resulting VSMCs carried persistent physiological defects compared to SKP-derived VSMC from subjects without T2D.

  • These data suggest utility of SKP-derived VSMCs as a model system for testing the vascular pathophysiology of T2D, and as a potential platform for personalized vascular medicine.

Funding

This study was funded by grants from the Canadian Institutes of Health Research (CIHR) [grant numbers MOP136850 and MOP14648 (to M.H.)]; The Heart and Stroke Foundation [grant number T6757 (to M.H.)]; and from the McEwen Centre for Regenerative Medicine (Acceleration Award) to M.H. S.K.S. was funded in part by a fellowship from the Heart and Stroke Richard Lewar Centre of Excellence.

Author contribution

S.K.S. conceived and designed the study, wrote the REB proposal, recruited the patients, designed the experiments, performed the experiments, and wrote the manuscript. T.M.Y. collected tissue samples. M.O. collected tissue samples. H.A.Q. performed statistical analysis. M.C. performed statistical analysis. T.K.W. collected tissue samples. M.H. conceived and designed the study, wrote the manuscript, and oversaw the study.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • AF

    atrial fibrillation

  •  
  • ASMA

    α-smooth muscle actin

  •  
  • AU

    arbitrary unit

  •  
  • AUC

    area under the curve

  •  
  • AVD

    aortic valve disease

  •  
  • CABG

    coronary artery bypass grafting

  •  
  • CAD

    coronary artery disease

  •  
  • CHF

    congestive heart failure

  •  
  • CKD

    chronic kidney disease

  •  
  • CVD

    cardiovascular disease

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • EGF

    epidermal growth factor

  •  
  • ESC

    embryonic stem cell

  •  
  • FGF2

    fibroblast growth factor 2

  •  
  • HBSS

    Hank’s balanced salt solution

  •  
  • HTN

    hypertension

  •  
  • iPSC

    induced pluripotent stem cell

  •  
  • MI

    myocardial infarction

  •  
  • MVD

    mitral valve disease

  •  
  • P1/P2

    passage 1/2

  •  
  • PAD

    peripheral arterial disease

  •  
  • PE

    phenylephrine

  •  
  • SKP

    skin-derived precursor

  •  
  • SmthnB

    smoothelin B

  •  
  • T2D

    type 2 diabetes

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TIA or CVA

    transient ischemic attack or cerebrovascular attack

  •  
  • VSMC

    vascular smooth muscle cell

References

References
1
Balkau
B.
,
Shipley
M.
,
Jarrett
R.J.
,
Pyorala
K.
,
Pyorala
M.
,
Forhan
A.
et al
(
1998
)
High blood glucose concentration is a risk factor for mortality in middle-aged nondiabetic men. 20-year follow-up in the whitehall study, the paris prospective study, and the helsinki policemen study
.
Diabetes Care.
21
,
360
367
2
Haffner
S.M.
,
Lehto
S.
,
Ronnemaa
T.
,
Pyorala
K.
and
Laakso
M.
(
1998
)
Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction
.
N. Engl. J. Med.
339
,
229
234
3
Aronson
D.
and
Rayfield
E.J.
(
2002
)
How hyperglycemia promotes atherosclerosis: Molecular mechanisms
.
Cardiovasc. Diabetol.
1
,
1
4
Go
A.S.
,
Mozaffarian
D.
,
Roger
V.L.
,
Benjamin
E.J.
,
Berry
J.D.
,
Borden
W.B.
et al
(
2013
)
Executive summary: Heart disease and stroke statistics–2013 update: A report from the American Heart Association
.
Circulation
127
,
143
152
5
Islam
M.S.
and
Loots du
T.
(
2009
)
Experimental rodent models of type 2 diabetes: a review
.
Methods Find. Exp. Clin. Pharmacol.
31
,
249
261
6
King
A.J.
(
2012
)
The use of animal models in diabetes research
.
Br. J. Pharmacol.
166
,
877
894
7
Kolodziejska
K.M.
,
Noyan-Ashraf
M.H.
,
Nagy
A.
,
Bacon
A.
,
Frampton
J.
,
Xin
H.B.
et al
(
2008
)
C-myb-dependent smooth muscle cell differentiation
.
Circ. Res.
102
,
554
561
8
Ishida
M.
,
El-Mounayri
O.
,
Kattman
S.
,
Zandstra
P.
,
Sakamoto
H.
,
Ogawa
M.
et al
(
2012
)
Regulated expression and role of c-myb in the cardiovascular-directed differentiation of mouse embryonic stem cells
.
Circ. Res.
110
,
253
264
9
El-Mounayri
O.
,
Mihic
A.
,
Shikatani
E.A.
,
Gagliardi
M.
,
Steinbach
S.K.
,
Dubois
N.
et al
(
2013
)
Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells
.
Cardiovasc. Res.
98
,
125
135
10
Fernandes
K.J.
,
McKenzie
I.A.
,
Mill
P.
,
Smith
K.M.
,
Akhavan
M.
,
Barnabe-Heider
F.
et al
(
2004
)
A dermal niche for multipotent adult skin-derived precursor cells
.
Nat. Cell Biol.
6
,
1082
1093
11
Toma
J.G.
,
Akhavan
M.
,
Fernandes
K.J.
,
Barnabe-Heider
F.
,
Sadikot
A.
,
Kaplan
D.R.
et al
(
2001
)
Isolation of multipotent adult stem cells from the dermis of mammalian skin
.
Nat. Cell Biol.
3
,
778
784
12
Toma
J.G.
,
McKenzie
I.A.
,
Bagli
D.
and
Miller
F.D.
(
2005
)
Isolation and characterization of multipotent skin-derived precursors from human skin
.
Stem Cells
23
,
727
737
13
Steinbach
S.K.
,
El-Mounayri
O.
,
DaCosta
R.S.
,
Frontini
M.J.
,
Nong
Z.
,
Maeda
A.
et al
(
2011
)
Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells
.
Arterioscler. Thromb. Vasc. Biol.
31
,
2938
2948
14
Tiscornia
G.
,
Vivas
E.L.
and
Izpisua Belmonte
J.C.
(
2011
)
Diseases in a dish: modeling human genetic disorders using induced pluripotent cells
.
Nat. Med.
17
,
1570
1576
15
Cherry
A.B.
and
Daley
G.Q.
(
2012
)
Reprogramming cellular identity for regenerative medicine
.
Cell
148
,
1110
1122
16
Dow
L.E.
and
Lowe
S.W.
(
2012
)
Life in the fast lane: mammalian disease models in the genomics era
.
Cell
148
,
1099
1109
17
Doi
A.
,
Park
I.H.
,
Wen
B.
,
Murakami
P.
,
Aryee
M.J.
,
Irizarry
R.
et al
(
2009
)
Differential methylation of tissue- and cancer-specific cpg island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts
.
Nat. Genet.
41
,
1350
1353
18
Lister
R.
,
Pelizzola
M.
,
Kida
Y.S.
,
Hawkins
R.D.
,
Nery
J.R.
,
Hon
G.
et al
(
2011
)
Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells
.
Nature
471
,
68
73
19
Kim
K.
,
Doi
A.
,
Wen
B.
,
Ng
K.
,
Zhao
R.
,
Cahan
P.
et al
(
2010
)
Epigenetic memory in induced pluripotent stem cells
.
Nature
467
,
285
290
20
Kim
K.
,
Zhao
R.
,
Doi
A.
,
Ng
K.
,
Unternaehrer
J.
,
Cahan
P.
et al
(
2011
)
Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells
.
Nat. Biotechnol.
29
,
1117
1119
21
Brennand
K.J.
(
2013
)
Inducing cellular aging: enabling neurodegeneration-in-a-dish
.
Cell Stem Cell
13
,
635
636
22
Wu
B.
,
Bell
K.
,
Stanford
A.
,
Kern
D.M.
,
Tunceli
O.
,
Vupputuri
S.
et al
(
2016
)
Understanding ckd among patients with t2dm: prevalence, temporal trends, and treatment patterns-nhanes 2007-2012
.
BMJ Open Diabetes Res. Care
4
,
e000154
23
Movahed
M.R.
,
Hashemzadeh
M.
and
Jamal
M.M.
(
2007
)
Significant increase in the prevalence of non-rheumatic aortic valve disease in patients with type 2 diabetes mellitus
.
Exp. Clin. Endocrinol. Diabetes
115
,
105
107
24
Movahed
M.R.
,
Hashemzadeh
M.
and
Jamal
M.M.
(
2005
)
The prevalence of pulmonary embolism and pulmonary hypertension in patients with type ii diabetes mellitus
.
Chest
128
,
3568
3571
25
Shah
A.D.
,
Langenberg
C.
,
Rapsomaniki
E.
,
Denaxas
S.
,
Pujades-Rodriguez
M.
,
Gale
C.P.
et al
(
2015
)
Type 2 diabetes and incidence of cardiovascular diseases: a cohort study in 1.9 million people
.
Lancet Diabetes Endocrinol.
3
,
105
113
26
Dickhuth
J.
,
Koerdt
S.
,
Kriegebaum
U.
,
Linz
C.
,
Muller-Richter
U.D.
,
Ristow
O.
et al
(
2015
)
In vitro study on proliferation kinetics of oral mucosal keratinocytes
.
Oral Surg. Oral Med. Oral Pathol. Oral Radiol.
120
,
429
435
27
Fleischhacker
E.
,
Esenabhalu
V.E.
,
Spitaler
M.
,
Holzmann
S.
,
Skrabal
F.
,
Koidl
B.
et al
(
1999
)
Human diabetes is associated with hyperreactivity of vascular smooth muscle cells due to altered subcellular Ca2+ distribution
.
Diabetes
48
,
1323
1330
28
Biernaskie
J.
,
Paris
M.
,
Morozova
O.
,
Fagan
B.M.
,
Marra
M.
,
Pevny
L.
et al
(
2009
)
Skps derive from hair follicle precursors and exhibit properties of adult dermal stem cells
.
Cell Stem Cell
5
,
610
623
29
Sato
H.
,
Ebisawa
K.
,
Takanari
K.
,
Yagi
S.
,
Toriyama
K.
,
Yamawaki-Ogata
A.
et al
(
2014
)
Skin-derived precursor cells promote wound healing in diabetic mice
.
Ann. Plast. Surg.
30
Libby
P.
(
1987
)
The active roles of cells of the blood vessel wall in health and disease
.
Mol. Aspects Med.
9
,
499
567
31
Schiffrin
E.L.
(
1994
)
Intracellular signal transduction for vasoactive peptides in hypertension
.
Can. J. Physiol. Pharmacol.
72
,
954
962
32
Endemann
D.H.
and
Schiffrin
E.L.
(
2004
)
Endothelial dysfunction
.
J. Am. Soc. Nephrol.
15
,
1983
1992
33
Wight
T.N.
and
Merrilees
M.J.
(
2004
)
Proteoglycans in atherosclerosis and restenosis: key roles for versican
.
Circ. Res.
94
,
1158
1167
34
Siow
R.C.
,
Sato
H.
and
Mann
G.E.
(
1999
)
Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide?
Cardiovasc. Res.
41
,
385
394
35
Faries
P.L.
,
Rohan
D.I.
,
Takahara
H.
,
Wyers
M.C.
,
Contreras
M.A.
,
Quist
W.C.
et al
(
2001
)
Human vascular smooth muscle cells of diabetic origin exhibit increased proliferation, adhesion, and migration
.
J. Vasc. Surg.
33
,
601
607
36
Oikawa
S.
,
Hayasaka
K.
,
Hashizume
E.
,
Kotake
H.
,
Midorikawa
H.
,
Sekikawa
A.
et al
(
1996
)
Human arterial smooth muscle cell proliferation in diabetes
.
Diabetes
45
,
S114
S116
37
Chung
A.W.
,
Luo
H.
,
Tejerina
T.
,
van Breemen
C.
and
Okon
E.B.
(
2007
)
Enhanced cell cycle entry and mitogen-activated protein kinase-signaling and downregulation of matrix metalloproteinase-1 and -3 in human diabetic arterial vasculature
.
Atherosclerosis
195
,
e1
e8
38
Madi
H.A.
,
Riches
K.
,
Warburton
P.
,
O’Regan
D.J.
,
Turner
N.A.
and
Porter
K.E.
(
2009
)
Inherent differences in morphology, proliferation, and migration in saphenous vein smooth muscle cells cultured from nondiabetic and type 2 diabetic patients
.
Am. J. Physiol. Cell Physiol.
297
,
C1307
C1317
39
Riches
K.
,
Alshanwani
A.R.
,
Warburton
P.
,
O’Regan
D.J.
,
Ball
S.G.
,
Wood
I.C.
et al
(
2014
)
Elevated expression levels of mir-143/5 in saphenous vein smooth muscle cells from patients with type 2 diabetes drive persistent changes in phenotype and function
.
J. Mol. Cell. Cardiol.
74
,
240
250
40
Riches
K.
,
Warburton
P.
,
O’Regan
D.J.
,
Turner
N.A.
and
Porter
K.E.
(
2014
)
Type 2 diabetes impairs venous, but not arterial smooth muscle cell function: possible role of differential rhoa activity
.
Cardiovasc. Revasc. Med.
15
,
141
148
41
Dhar
G.C.
(
2009
)
Intensive glycemic control: Implications of the accord, advance, and vadt trials for family physicians
.
Can. Fam. Physician
55
,
803
804
42
Brem
H.
,
Sheehan
P.
,
Rosenberg
H.J.
,
Schneider
J.S.
and
Boulton
A.J.
(
2006
)
Evidence-based protocol for diabetic foot ulcers
.
Plast. Reconstr. Surg.
117
,
193S
209S
43
Jeffcoate
W.J.
and
Harding
K.G.
(
2003
)
Diabetic foot ulcers
.
Lancet
361
,
1545
1551