Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture - PubMed (original) (raw)

Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture

Maryam Masouminia et al. J Sex Med. 2018 Dec.

Abstract

Background: Previous work showed that muscle-derived stem cells (MDSCs) exposed long-term to the milieu of uncontrolled type 2 diabetes (UC-T2D) in male obese Zucker (OZ) rats, were unable to correct the associated erectile dysfunction and the underlying histopathology when implanted into the corpora cavernosa, and were also imprinted with a noxious gene global transcriptional signature (gene-GTS), suggesting that this may interfere with their use as autografts in stem cell therapy.

Aim: To ascertain the respective contributions of dyslipidemia and hyperglycemia to this MDSC damage, clarify its mechanism, and design a bioassay to identify the damaged stem cells.

Methods: Early diabetes MDSCs and late diabetes MDSCs were respectively isolated from nearly normal young OZ rats and moderately hyperglycemic and severely dyslipidemic/obese aged rats with erectile dysfunction. Monolayer cultures of early diabetic MDSCs were incubated 4 days in DMEM/10% fetal calf serum + or - aged OZ or lean Zucker serum from non-diabetic lean Zucker rats (0.5-5%) or with soluble palmitic acid (PA) (0.5-2 mM), cholesterol (CHOL) (50-400 mg/dL), or glucose (10-25 mM).

Main outcome measure: Fat infiltration was estimated by Oil red O, apoptosis by TUNEL, protein expression by Western blots, and gene-GTS and microRNA (miR)-GTS were determined in these stem cells' RNA.

Results: Aged OZ serum caused fat infiltration, apoptosis, myostatin overexpression, and impaired differentiation. Some of these changes, and also a proliferation decrease occurred with PA and CHOL. The gene-GTS changes by OZ serum did not resemble the in vivo changes, but some occurred with PA and CHOL. The miR-GTS changes by OZ serum, PA, and CHOL resembled most of the in vivo changes. Hyperglycemia did not replicate most alterations.

Clinical implications: MDSCs may be damaged in long-term UC-T2D/obese patients and be ineffective in autologous human stem cell therapy, which may be prevented by excluding the damaged MDSCs.

Strength & limitations: The in vitro test of MDSCs is innovative and fast to define dyslipidemic factors inducing stem cell damage, its mechanism, prevention, and counteraction. Confirmation is required in other T2D/obesity rat models and stem cells (including human), as well as miR-GTS biomarker validation as a stem cell damage biomarker.

Conclusion: Serum from long-term UC-T2D/obese rats or dyslipidemic factors induces a noxious phenotype and miR-GTS on normal MDSCs, which may lead in vivo to the repair inefficacy of late diabetic MDSCs. This suggests that autograft therapy with MDSCs in long-term UT-T2D obese patients may be ineffective, albeit this may be predictable by prior stem cell miR-GTS tests. Masouminia M, Gelfand R, Kovanecz I, et al. Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture. J Sex Med 2018;15:1678-1697.

Keywords: Apoptosis; Cholesterol; Diabetes Control; Erectile Dysfunction; Fat Infiltration; Free Fatty Acids; MicroRNA; Muscle-Derived Stem Cells; Myostatin; Palmitic Acid; Peyronie’s Disease; miR.

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Figures

Figure 1.. A moderate hyperglycemia but intense dyslipidemia develops with age in male diabetic obese Zucker (OZ) rats in contrast to the related strain non-diabetic lean Zucker (LZ) rats.

Animals were subjected to weekly evaluation of glucose, cholesterol, triglycerides, and insulin (not shown) in the serum from 12 to 28 weeks of age.

Figure 2.. Incubation of ED-MDSC with a higher level of aged OZ serum induced a considerable intracellular infiltration by fat globules.

ED-MDSC were incubated with no addition (A), or with added 5% LZ serum (B) or 5% OZ serum (C) as in Fig. 2 Supplement, and stained with Oil Red O. Pictures were taken at 200X, but QIA was applied to multiple fields at 100X (D) Graph of red area (fat) per cell. ***p≤0.005 (CTR vs OZ); ○○○p≤0.005 (LZ vs OZ)

Figure 3.. Incubation of ED-MDSC with water-soluble preparations of the sodium salt of palmitic acid (PA) as representative of saturated free fatty acids, or cholesterol (CHOL), induced a milder infiltration of fat globules.

ED-MDSC were incubated with no addition (A), or with added PA at 0.5 (B) or 1 mM (not shown), or added CHOL at 50 mg/dl (C), or 100 mg/dl (not shown) and stained with Oil Red O. Pictures were taken at 200X, but QIA was applied to multiple fields at 100X (D) Graph of red area (fat) per cell. ***p≤0.005 (CTR vs OZ)

Figure 4.. Incubation of ED-MDSC with 5% aged OZ serum induced moderate apoptosis.

ED-MDSC were incubated with no addition (A), or with added 5% serum from aged LZ rats (B) or 5% serum from aged OZ rats (C) as in Fig. 2 and stained with the Tunel reaction. Pictures were taken at 200X, but QIA was applied to multiple fields at 100X (D). Graph of apoptotic index. ***p≤0.005 (CTR vs OZ); ○○p≤0.01 (LZ vs OZ) The morphology of the control cells appears slightly different than in the previous figures stained for Oil Red O, but leaving aside the typical stem cell coexistence of morphological variants, is the different background color in both types of reactions what mostly creates this impression.

Figure 5.. Incubation of ED-MDSC with preparations of water-soluble palmitic acid (PA), or cholesterol (CHOL), induced considerable apoptosis.

ED-MDSC were incubated with no addition (A), or with added PA at 0.5 (B), or added CHOL at 50 mg/dl (B) or 100 (C) and stained with the Tunel reaction. Pictures were taken at 200X, but QIA was applied to multiple fields at 100X (D) Graph of apoptotic index. ***p≤0.005 (CTR vs OZ); ○○○p≤0.005 (LZ vs OZ)

Figure 6.. Incubation of ED-MDSC with OZ serum caused a moderate decrease in cell replication and smooth muscle differentiation.

ED-MDSC were incubated in duplicate with no addition, with added 0.5%, 2.5%, or 5% aged OZ or LZ serum, and subjected to western blot as indicated, with beta-actin as a housekeeping gene. See Fig. 7 for quantitative analysis.

Figure 7.. The quantitative image analysis (QIA) of the protein band densitometries in the incubations of ED-MDSC with OZ serum and palmitic acid sodium salt confirmed the visual inspection of the western blot images.

The selected bands of the duplicate experiments seen in Fig. 6 and in Fig. 2 Supplement (single experiment with cholesterol excluded) were subjected to QIA and the means+/−SEM were statistically compared for variance in each type of incubations. *p≤0.05 compared to control

Figure 8.. Incubation of ED-MDSC with increasing aged OZ serum caused a concentration dependent expression of myostatin protein, a pro-lipofibrotic and muscle mass inhibitor, but the incubation with increasing glucose exerted an opposite effect.

Incubations were performed on 6 well plates with no addition, or in duplicate with increasing concentrations of OZ or LZ serum (A), or of glucose either before or after 2 mM azacytidine for 2 days to stimulate stemness (C). Cell homogenates were subjected to western blot as indicated, with beta-actin as housekeeping gene, and densitometry was applied for the A samples (B). Other biomarkers for fibrosis, apoptosis, replication, and SMC differentiation were assayed in C, but no changes were observed (not shown). *p≤0.05 compared to control

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Dyslipidemia Is a Major Factor in Stem Cell Damage Induced by Uncontrolled Long-Term Type 2 Diabetes and Obesity in the Rat, as Suggested by the Effects on Stem Cell Culture - PubMed (original) (raw)