Hyperglycemic conditions induce phenotypic changes in human aortic SMCs

Significant differences in morphology and phenotype were evident in SMCs exposed to various glucose conditions (Fig. 1, A, B). It was noted that cells were more aggregated, irregular in shape, and larger in cross-sectional area within high glucose cultures, contrasting with the regular and standalone appearance in 5 mmol/L cultures. T2DM cells lacked the hill and valley configuration seen in normal SMCs, in line with that reported by others [34]. Analysis of the cell morphologies from multiple such images revealed that the cell area and perimeter significantly increased at higher glucose concentration (Fig. 1, C; p < 0.05), whereas T2DM cells were the largest in size (p < 0.05) among all the cases. This is to be expected because high glucose levels reportedly have differential effects on the phenotype and function of many cell types. The cell morphology was analyzed using the cell shape index, i.e., \(\:CSI\:=\:4\pi\:A/^\), where A and P are the measured cell area and perimeter, respectively [35]. Results indicated that SMCs exposed to 10 mmol/L and 20 mmol/L glucose had CSI around 0.23 ± 0.01, whereas T2DM cells and normal SMCs (5 mmol/L) had CSI around 0.48 ± 0.03 (p < 0.001). It should be noted that the area and perimeter of the SMCs in control (5 mmol/L glucose) cultures is in broad agreement with literature [36]. Quantitative analysis revealed elevated levels of F-actin expression in SMCs receiving higher glucose concentrations (Fig. 1, D, E). The area covered by actin within the cells as well as the fluorescence intensity of actin were significantly higher in 20 mmol/L glucose treated cells and T2DM-SMCs compared to cells receiving 5 mmol/L or 10 mmol/L glucose (p < 0.05). Collectively, these results suggest that exposure of VSMCs to elevated glucose levels for longer periods induces significant phenotypic changes in these cells, broadly comparable to their T2DM counterparts.

The Young’s modulus (EY) of SMCs in normal glucose conditions (5 mmol/L) was noted as 7.03 ± 2.01 kPa (Fig. 1, F). While 10 mmol/L glucose did not contribute to significant changes in EY (7.78 ± 1.65 kPa; p > 0.05 vs. 5 mmol/L), 20 mmol/L glucose significantly reduced EY to 3.32 ± 1.3 kPa (p < 0.05 vs. 5 mmol/L; p < 0.05 vs. 10 mmol/L glucose). T2DM cells exhibited EY (3.19 ± 0.84 kPa) similar to SMCs that received 20 mmol/L (p < 0.01 vs. 5 mmol/L; p < 0.01 vs. 20 mmol/L). The EY of SMCs in 5 mmol/L glucose conditions was similar to our previous study [37], whereas the EY of T2DM-SMCs is in close range to values reported in literature [38]. Human coronary SMCs derived from healthy patients reportedly had higher EY than those isolated from T2DM patients [38], in agreement with our results here. However, in coronary vascular SMC cultures, isolated from control or diabetic mice, no significant impact of glucose concentration on EY was reported [38]. SMCs receiving 5 mmol/L glucose recorded adhesion forces (Fad) of 558 ± 229 pN (Fig. 1, G), and increasing the glucose concentration significantly decreased these adhesion forces (p < 0.05 vs. 5 mmol/L; p < 0.05 for 10 mmol/L vs. 20 mmol/L). T2DM cells had the lowest adhesion forces of all the cases tested (233 ± 71 pN; p < 0.05 vs. all other cases). The tether forces (FT) recorded in SMCs cultured with 5 mmol/L glucose was 787 ± 342 pN (Fig. 1, H), and while 10 mmol/L glucose had no effect on these values, 20 mmol/L significantly reduced the FT (286 ± 109 pN; p < 0.05 vs. 5 mmol/L and 10 mmol/L). T2DM cells recorded the lowest FT among all the test cases and was not significantly different from 20 mmol/L condition.

The force needed to deform a cell membrane, also termed as the apparent membrane tension (TM), was calculated from tether forces using \(\:_\cong\:_^/8^_\), where KB is the bending stiffness that lies in the 0.1–0.3 pN.µm range [39, 40]. The TM values were as follows (Fig. 1, I): 5 mmol/L (50.4 ± 2.3 nN/µm), 10 mmol/L (53.7 ± 3.2 nN/µm), 20 mmol/L (11.4 ± 0.9 nN/µm), and T2DM (3.5 ± 0.2 nN/µm). The TM was significantly lower in cells receiving 20 mmol/L glucose versus other glucose concentrations (p < 0.01), and T2DM cells have the lowest TM of all the cases tested (p < 0.01). The tether radius (RT) increased with increasing glucose concentration (Fig. I, J), with control (5 mmol/L) cells having 1.9 ± 0.03 nm on average and T2DM cells having 6.1 ± 0.13 nm. To the best of our knowledge, the FT, Fad, TM and RT values for human SMCs and specifically under diabetic conditions haven’t been reported earlier in literature.

Previously, we reported that primary SMCs derived from human aneurysmal aortae and analyzed using AFM exhibited the following values [41]: EY = 20.9 ± 7.7 kPa, Fad = 1.87 ± 0.13 nN, FT = 218.8 ± 14.3 pN, TM = 6.07 ± 0.8 nN/µm, and RT = 2.91 ± 0.19 nm. Compared to T2DM SMCs and SMCs receiving higher glucose in the current study, it is evident that human aneurysmal aortic SMCs (yet another CVD condition) have significantly higher EY, Fad and RT (p < 0.01 in all the cases), although the tether forces and membrane tension of T2DM SMCs were comparable to that in aneurysmal SMCs, underlying the differences in the pathologies of these two vascular diseases.

Compared to SMCs cultured for 24 h, cell proliferation increased 1.3–1.75 −fold after 48 h, 4.1–5.35 −fold after 96 h, and 5.5–6.1 −fold after 7 days (Fig. 1, K) in all the cases. Cell proliferation increased with time at each glucose concentration, as well as in T2DM cultures. Our findings are consistent with previous reports where high glucose led to significant increase in the proliferation of various cell types compared to normal glucose levels [42]. For instance, previous studies reported a 3−fold increase in VSMC proliferation exposed to high glucose (> 20 mmol/L) compared to controls (5 mmol/L), as measured using the MTT assay [43,44,45].

Hyperglycemia has been linked to numerous facets of arterial modeling. Elevated glucose levels have been shown to alter trophoblast proliferation and invasion, thereby contributing to SMC mediated arterial remodeling during fetal growth and development [46]. T2DM patients with higher glucose levels had larger common carotid artery intima-media thickness, luminal diameter, and brachial pulse pressure compared to their healthy counterparts, suggesting adaptive remodeling of stiffened arteries [47]. Clinically, T2DM has been implicated in remodeling of calcified arteries that exhibit significantly greater necrotic cores and inflammation, primarily driven by macrophages and lymphocytes [48]. Hyperglycemia induces significant changes in vascular ECM composition and remodeling capabilities, ultimately leading to compromised vessel function and stiffer vessel walls [49]. A majority of these changes are driven by alternations in SMC phenotype, function, and mechanobiology, resulting in diseases such as stroke, aneurysm, and hypertensive vessels [16, 50].

Fig. 1

A. Representative phase-contrast images of adult human aortic SMCs cultured under varying glucose concentrations (5, 10, or 20 mmol/L), and T2DM SMCs. B. Representative confocal images of filamentous-actin (F-actin) and nuclei (blue) stained with Alexa Fluro 488-phalloidin and DAPI, respectively. C. Significant differences in average cell area and perimeter were noted between SMCs treated with various dosages of glucose and with T2DM SMCs. Data shown represents mean ± standard error in respective cases (n > 50 cells/condition). **** indicates p < 0.0001 vs. 5 mmol/L cultures; #### indicates p < 0.0001 vs. 10 mmol/L cultures; ++++ indicates p < 0.0001 vs. 20 mmol/L cultures. D. Quantitative analysis of area occupied by F-actin within SMCs exposed to various culture conditions. E. Quantitative analysis of fluorescence intensity of F-actin. Analysis of fluorescence intensity was done at the original magnification by measuring the mean gray value with Fiji ImageJ software. Data was pooled from n = 6 wells/ condition for each of these assays. Biomechanical characteristics such as elastic modulus (EY, F), forces of adhesion (Fad, G), membrane tether forces (FT, H), membrane tension (TM, I), and tether radius (RT, J) were quantified from the AFM data. EY data were calculated by applying Hertz model to force–indentation curves (n ≥ 100 cells/condition) obtained from cells. Fad and FT were measured by retraction of beaded-AFM probe from the cell surface. In plots D-J, the center line in the box plots denotes the median, and bound of box shows 25th to 75th percentiles, while upper and lower bounds of whiskers represent the maximum and minimum values, respectively. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. K. Proliferation of human SCMs under various glucose conditions as well as that of T2DM-SMCs. * indicates p < 0.05 vs. initial cell seeding density, ## indicates p < 0.01 for control (5 mmol/L) vs. glucose treatment (10 mmol/L, 20 mmol/L; T2DM)

Differential effects of hyperglycemia on cytokine/ chemokine/ growth factor release by SMCs

The release of cytokines and chemokines in chronic SMC cultures were quantified (Fig. 2) and significant variations in analyte concentrations depending on glucose concentration were noted. Some interleukins that weren’t expressed at all or released in extremely low levels by T2DM cells (e.g., IL-9, IL-17 A, IL-17 F, IL-28 A, IL-4, IL-15, IL-25, IL-33) were seen in SMC cultures receiving glucose (Fig. 2, A). On the other hand, interleukins such as IL-6, IL-8, IL-23 and IL-27 were released in higher levels in T2DM cells than in SMCs receiving glucose. Among various cytokines and chemokines tested (Fig. 2, B), higher levels of SCD40L, RANTES, GROα, MCP-1, M-CSF, and MIP-1δ were noted in T2DM cultures than in SMC cultures receiving glucose. On the other hand, G-CSF was higher in SMC cultures receiving glucose than in T2DM cultures. There weren’t any significant differences in the expression of other cytokines and chemokines between the various culture conditions. T2DM has been linked to immune system disorders as well as elevated levels of IL-6, IL-18, MCP-1, and TNF-α [51,52,53]. Although TNF-α, IL-18, and IFN-γ levels were similar in all the cases we tested, IL-6 and MCP-1 levels were significantly higher in T2DM cells consistent with literature.

FGF-2 levels were slightly higher in SMC cultures receiving 5 and 10 mmol/L glucose, whereas TGF-β2 and TGF-β3 levels were significantly lower in T2DM cultures (Fig. 2, C). The levels of VEGF-A, PDGF-AA, and TGF-β1 were higher in cultures receiving 10 and 20 mmol/L glucose. Similar to our observation, high glucose (20 mmol/L) was shown to elevate TGF-β1 and TGF-β-R1 receptor expression in vascular SMCs, via protein kinase C (PKC-α) activation [54]. Hyperglycemia was shown to induce VEGF-A expression in SMCs similar to acute insulin treatment [55], with implications in decreased function and failure of multiple organs (e.g., retina, kidneys). Our study showed that VEGF-A levels were significantly elevated (> 40-fold; p < 0.001 vs. 5 mmol/L) in the presence of 10 mmol/L and 20 mmol/L glucose to levels noted in T2DM cells.

MMPs–1, 2, 3 and TIMPs–1, 2, 3 were elevated in T2DM cells than glucose-receiving HASMC cultures (Fig. 2, D). These results suggest that SMCs treated with varying glucose concentrations directly influence the expression of various cytokines, chemokines, growth factors and MMPs/ TIMPs. High glucose exposure was shown to increase transcription and translation of MMPs−1, 2, 9 and 13 in human SMC cultures and their enzymatic activity, especially in the presence of macrophages, representative of T2DM conditions in vivo [56]. Consistent with this report, we here note that these specific MMPs were significantly higher in T2DM cultures, whereas MMPs−2, 9 and 12 were also present in SMC cultures receiving high glucose levels. The interplay between MMPs and TIMPs regulate the development of atherosclerotic plaques and arterial remodeling under diabetic conditions [57]. In humans with T2DM and hypertension, elevated levels of TIMPs-1 and 3, TIMP-1: MMP-2 ratio, and TIMP-1: MMP-9 ratio were reported [58, 59], which mirrors our observations in this study.

Fig. 2

Heat maps of the levels of interleukins (A), cytokines & chemokines (B), growth factors (C), and MMPs/TIMPs (D) released in SMC cultures receiving various glucose concentrations and from T2DM cells over 21 days. Spent media was pooled from at least n = 6 wells/ condition and processed to measure the levels of these markers

It is worth noting that previous studies suggest that when human aortic SMCs were co-cultured with macrophages under normal (5.5 mmol/L) and high glucose (25 mmol/L) conditions, SMCs exhibited augmented gene and protein expression of MMP-1 and MMP-9, significant increase in MMP-9 enzymatic activity, higher levels of soluble and functionally-active MCP-1 linked to MMPs upregulation, and activated PKCα signaling pathway that together with NF-kB is responsible for MMPs-1, 9 upregulation [56].

Inflammatory cytokines-associated pathways are enriched in hyperglycemia induced upregulated genes

Previous research has independently analyzed the transcriptome, proteomics, and metabolomics to identify distinct markers in diabetic SMCs [60]. Our current study, however, profiles these as paired omics data. We explored which genes correspond with metabolomics profiling patterns and the implications of these correlations within the omics modules. We also examined how mRNA levels align with protein abundance. This integrated dataset analysis deepens our understanding of chronic hyperglycemia-induced changes in human aortic smooth muscle cells, advancing beyond mere biomarkers to explore their complex relationships.

Fig. 3

Volcano plots of differential gene expression patterns and enriched pathways in response to glucose concentration and diabetic conditions. (A) 10 mmol/L vs. 5 mmol/L glucose, (B) 20 mmol/L vs. 5 mmol/L glucose, and (C) T2DM Vs 5 mmol/L glucose. Cell pellets were pooled from n = 6 wells/ condition for RNA-seq analysis

We first investigated the genes upregulated by hyperglycemia enriched pathways. Vascular SMCs receiving 5 mmol/L glucose were compared to those receiving 10 mmol/L (Fig. 3, A) or 20 mmol/L (Fig. 3, B) glucose, and T2DM-SMCs (Fig. 3, C). Pathway enrichment analysis was further performed by the differentially expressed genes (DEGs). We found that inflammatory cytokine-associated pathways, especially those involving IL-10 signaling, are prominently enriched in genes that are up-regulated by increased glucose levels of 10 mmol/L and 20 mmol/L glucose, compared to normal glucose levels. IL-10 signaling pathway has been shown to modulate the function of various immune cells. Studies have shown that elevated IL-10 pathways are associated with multiple autoimmune diseases [35]. Additionally, we observed that genes activated by a higher glucose concentration of 20 mmol/L are more closely associated with inflammatory cytokine pathways, including not only IL−10 but also ILs − 1, 4 and 13 signaling pathways (Fig. 3, B). This aligns with the hypothesis that long-term exposure to high glucose levels leads to enhanced inflammatory responses, which are associated with chorionic inflammation. The genes involved in the activation of G-alpha signaling events were significantly enriched in SMCs receiving 20 mmol/L glucose but not in SMCs supplied with 10 mmol/L glucose. G-alpha is involved in the inhibition of cAMP dependent pathway which in turn leads to reduced activity of cAMP-dependent protein kinases, as well as activation of the protein tyrosine kinase c-Src [61, 62].

The epidermal growth factor receptor family (EGFR or ErbB 1−4) has been implicated in various cellular functions (e.g., growth, division, differentiation, migration, apoptosis) and multiple downstream signaling pathways (e.g., ERK1/2, MAP, PI3-kinase/Akt) under hyperglycemia or diabetes (types I and II) conditions and related cardiovascular outcomes [63]. In our study, compared to normal glucose levels, chronic exposure to 10 mmol/L but not 20 mmol/L glucose appears to have resulted in differentially expressed genes related to ErbB2 and ErbB4 and their functions and pathways (cell motility, PI3K, PTK6, SHC1) in human vascular SMCs.

We found a notable distinction between the gene expression patterns in cells exposed to high glucose levels in vitro (10 or 20 mmol/L) and those compared to T2DM-SMCs (Fig. 3, C). Despite some similarities, the up-regulated genes in T2DM cells did not entirely correspond with those observed in the high glucose in vitro models. This could possibly be due to the multifactorial nature of T2DM in vivo and exposure of SMCs to chronically (multi-year) elevated levels of high glucose and inflammatory molecules under such conditions. In T2DM-SMCs, the dominant pathways involved relate to ECM organization (e.g., laminin, elastin, collagens, proteoglycans, GAGs) and the regulation of IGF, highlighting the complexity of diabetes pathogenesis beyond the changes induced by hyperglycemia alone. This is to be partially expected because T2DM diagnosis could also be an indicator of the onset of various proteolytic vascular conditions such as atherosclerosis that results in arterial remodeling and SMC activation, which is reflected in the cells isolated from a T2DM tissue. Interestingly, IL-10 pathways were enriched in both the in vitro models and in T2DM-SMCs, suggesting that increased inflammatory cytokine-associated pathways are a consistent feature of prolonged hyperglycemia. This could potentially contribute to the pathogenesis of diabetic complications, particularly in vascular tissues, implying that controlling inflammation may be crucial in preventing these complications. Interestingly, compared to SMCs receiving normal glucose levels, T2DM cells had altered gene expression for molecules involved in ECM organization, elastic fiber synthesis and formation, laminin interactions, and ECM proteoglycans, highlighting the role of T2DM in arterial remodeling.

Fig. 4

Venn diagram indicating the overlap between differentially expression genes (DEG) in SMCs receiving various glucose concentrations (10 mmol/L and 20 mmol/L). RNA-seq data for each condition was compared to cells receiving 5 mmol/L glucose. T2DM cells were also shown for comparison. (A) Up-regulated differentially expressed genes; (B) down-regulated differentially expressed genes. Cell pellets were pooled from n = 6 wells/ condition for RNA-seq analysis

Similar outcomes were noted from the differentially regulated gene (DGE) analysis (Fig. 4) performed on these cells. We performed statistical analysis on the normalized read count data to assess quantitative changes in expression levels between the various groups. Compared to SMCs receiving normal glucose levels, 513 genes were upregulated, and 590 genes were downregulated in T2DM cells (list of genes provided in Table S1). We note that, among others, the genes involved in arterial remodeling were significantly upregulated in T2DM cells (e.g., COL, ELN, GLB, FBLN, LMN). We performed a ReactomePA analysis to identify the pathways enriched for the upregulated and downregulated genes, respectively). Cells receiving 10 and 20 mmol/L glucose have much fewer genes that were differentially expressed, while very few of these genes were common between all the cell types.

In the medial layers of healthy vascular ECM, circumferential layers of collagens, elastic fiber associated proteins (elastin, fibrillin, fibulins, microfibril-associated glycoproteins), as well as basement membrane proteins (collagen type IV, laminins, fibronectin) are present [64]. The signaling from healthy collagens and elastic fibers maintain vascular SMCs in a quiescent, differentiated state and contributes to normal SMC functions. Vascular injuries and diseases such as T2DM induce changes in the tissue ECM (e.g., proteolysis of ECM proteins) and activates and dedifferentiates SMCs to a more proliferative and invasive phenotype, disturbing the ECM protein-SMC signaling pathways [49]. Hyperglycemic conditions were shown to increase osteogenic genes expression in vascular SMCs, and enhance the stiffness of arterial elastin and glycation of elastic fibers, leading to arterial calcification associated with diabetes [65]. Others reported that hyperglycemia and T2DM conditions significantly alter the expression of ECM genes such as elastin, fibulin, laminin, and collagen [66,67,68,69], which in turn contribute to undesirable outcomes in arterial remodeling. Our results indicate upregulation of COL, ELN, GLB, FBLN, LMN genes in T2DM cells, highlighting their role in disease pathogenesis and arterial remodeling. Upregulation of these genes could lead to significant increases in their protein synthesis and deposition, which contributes to ECM remodeling, cell-ECM interactions, and cell migration.

Using a microarray dataset (GSE26168) from the Gene Expression Omnibus database, Zhu et al.. identified 981 DEGs, of which 301 were upregulated and the rest downregulated [70]. These DEGs were highly enriched in cell differentiation, cell adhesion, intracellular signal transduction, and regulation of protein kinase activity, as well as cAMP signaling pathway, Rap1 signaling pathway, regulation of lipolysis in adipocytes, PI3K-Akt signaling pathway, and MAPK signaling pathway. Based on the PPI network of these DEGs, the top 6 genes contributing to T2DM initiation, progression, and intervention strategy were identified as PIK3R1, RAC1, GNG3, GNAI1, CDC42, and ITGB1. While none of these specific genes were in measurable levels in our cultures, GNG2, CDC20, CDCP1, and ITGB8 were upregulated, whereas PIK3IP1 and ITGB3 were downregulated in T2DM cells. However, despite differences in the individual gene signatures between the two datasets and the underlying approaches (microarray in their study vs. RNA-seq here), pathway analysis highlights broad consistencies between our data and that reported by Zhu et al.

Significant correlations between mRNA and protein expression levels

We conducted a comparative analysis (Figs. 5 and 6) to understand the relationship between mRNA and protein expression levels by comparing gene expression data obtained from RNA-seq with protein levels determined through mass spectrometry (MS). Proteins were isolated from the extracellular matrix that was synthesized by SMCs and deposited in the culture wells (ECM), as well as from the cytosol (C) and cytoskeleton (CS) of cells. We observed a moderate yet statistically significant correlation between mRNA abundance as estimated by RNA-seq, and protein abundance as determined from MS. This correlation was quantified using Spearman’s correlation coefficient (Rho), which ranged from 0.24 to 0.6 across various samples. These findings suggest that alterations at the transcriptional level are broadly reflected at the protein level, influencing biological pathways.

The correlation between mRNA and protein abundance has been widely debated, as it varies significantly depending on the biological context. Studies have demonstrated that the correlation between mRNA and protein under specific conditions can range from 0.09 to 0.57, based on Spearman’s rank correlations [71]. In our data, the Spearman’s rank correlation between mRNA and protein ranges from 0.2 to 0.6, which is consistent with this typical range. While the relationship between mRNA and protein is context-dependent and complex, it does not necessarily mean that mRNA findings cannot inform protein levels. Rather, mRNA and proteins represent distinct regulatory layers, and protein abundance can be inferred from mRNA levels – not through direct correlation but using machine learning models that account for these intricate relationships [72].

Fig. 5

Correlation between ECM proteins and mRNA levels across various glucose conditions and T2DM patient samples for 448 genes. (A) 5 mmol/L: Spearman correlation coefficient of 0.438 (p = 1.67 × 10− 21). (B) 10 mmol/L: Spearman correlation coefficient of 0.453 (p = 4.11 × 10− 23). (C) 20 mmol/L: Spearman correlation coefficient of 0.439 (p = 1.53 × 10− 21). (D) T2DM: Spearman correlation coefficient of 0.533 (p = 7.55 × 10− 33). Cell pellets were pooled from n = 6 wells/ condition for RNA-seq and proteomic analysis

Fig. 6

The correlation between mRNA expression levels and cytosol (C) / cytoskeletal (CS) protein levels for 2323 genes across different culture conditions. (A) 5 mmol/L glucose: Spearman correlation coefficient of 0.245 (p = 2.67 × 10− 33) for cytosol protein. (B) 5 mmol/L condition: Spearman correlation coefficient of 0.343 (p = 3.71 × 10− 65) for cytoskeleton protein. (C) 10 mmol/L: Spearman correlation coefficient of 0.363 (p = 2.61 × 10− 72) for both cytosol (C) and cytoskeleton (CS) protein. (D) 20 mmol/L: Spearman correlation coefficient of 0.371 (p = 7.58 × 10− 77) for both cytosol (C) and cytoskeleton (CS) protein. (E) T2DM: Spearman correlation coefficient of 0.461 (p = 1.19 × 10− 122) for cytosol protein. (F) T2DM patient samples: Spearman correlation coefficient of 0.606 (p = 7.57 × 10− 234) for cytoskeleton protein. Cell pellets were pooled from n = 6 wells/ condition for RNA-seq and proteomic analysis

Identifying metabolites co-expressed genes and enriched pathways

Gene expressions and metabolites constitute two distinct layers of features that can characterize dynamic responses to changes in glucose levels. We investigated which genes are co-expressed with metabolites. For each metabolite, we identified gene sets that exhibited similar dynamic pattern