Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

doi:10.21203/rs.3.rs-3210647/v1

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Research Article

Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

https://doi.org/10.21203/rs.3.rs-3210647/v1

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published 24 Apr, 2024

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Background: Diabetic cardiomyopathy (DCM) is a serious health-threatening diabetic complication characterized by myocardial fibrosis and abnormal cardiac function. Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are considered as a potential therapeutic tool for DCM and myocardial fibrosis via the regulation of microRNA (miRNA) expression. This study aimed to investigate the therapeutic effects of tail vein injection of hUC-MSCs on DCM and determine effects on miRNA and target mRNA expression.

Methods: A DCM mouse model was induced by multiple low-dose streptozotocin (STZ) injections and the effect of hUC-MSCs administration was assessed at two time points, 10 and 18 weeks after induction of diabetes mellitus. Analysis of mouse heart tissues was undertaken two weeks after tail vein injection of hUC-MSCs. Biochemical methods, echocardiography, histopathology and polymerase chain reaction (PCR) were used to analyze blood glucose, body weight, cardiac structure and function, degree of myocardial fibrosis, and expression of fibrosis related mRNA and miRNA.

Results: DCM animals treated with saline had impaired cardiac function, increased fibrosis and decreased expression of miRNA-133a after 10 and 18 weeks of DM. The myocardial fibrosis and cardiac dysfunction induced in DCM mice were significantly improved 2 weeks after hUC-MSCs treatment at both an early and late disease time point. Furthermore, pro-fibrotic indicators such as α-SMA, collagen I, collagen III, Smad3, and Smad4 levels were significantly reduced after hUC-MSCs infusion compared with DCM hearts from animals treated with normal saline, and anti-fibrotic indicators such as FGF1 and miRNA-133a were significantly increased.

Conclusion: These results suggest that hUC-MSCs may improve cardiac function and myocardial fibrosis in DCM by regulating miRNA-133a and fibrosis related mRNAs.

Diabetic cardiomyopathy

Human umbilical cord mesenchymal stromal cells

miRNA-133a

Myocardial fibrosis

Fibrosis-related mRNA

Diabetes mellitus (DM) is a metabolic disease resulting in disordered glucose metabolism due to absolute or relative insufficiency of insulin secretion and/or insulin resistance [1]. The prevalence of DM is increasing rapidly worldwide. Among its many adverse effects, DM has been recognized as an independent risk factor for heart failure (HF) [2]. Diabetic cardiomyopathy (DCM), one of the target-organ complications of DM, is characterized by abnormal cardiac systolic and diastolic function, myocardial fibrosis and hypertrophy [3–6]. Myocardial fibrosis, an important pathological feature of DCM, is associated with glycosylated collagen deposition as a result of a prolonged hyperglycemic state [7]. MicroRNAs (miRNAs) are small non-coding RNAs with a length of 18–22 nucleotides, which can regulate protein expression at the mRNA level [8]. Alleviation of DCM and myocardial fibrosis by hUC-MSCs may be achieved by regulating the expression of miRNAs [9]. In addition, myocardial fibrosis is usually accompanied by changes in the intra-cardiac expression of a range of fibrosis-related gene products, such as alpha smooth muscle actin (αSMA), transforming growth factor beta (TGF-β), collagen I, collagen III, suppressor of mothers against decapentaplegic (SMAD)2, SMAD3, SMAD4 and fibroblast growth factor (FGF)1 [10].

Mesenchymal stromal cells (MSCs) originate from mesoderm and ectoderm in the early development process, and have multi-lineage differentiation potential [11]. Human umbilical cord MSCs (hUC-MSCs) are multifunctional cells that exist in neonatal umbilical cord tissue and have potential for broad clinical applications [12]. It is reported that hUC-MSCs have important anti-apoptotic, anti-fibrotic and anti-inflammatory properties in heart diseases, including DCM [13] [14]. However, the optimal timing of therapy and the primary mechanisms of action responsible for the prevention of myocardial fibrosis by systemic administration of hUC-MSCs in the setting of diabetes remain incompletely investigated.

The aim of the current study was to evaluate the effects of single intravenous (i.v.) injections of hUC-MSCs on cardiac function and myocardial fibrosis in a mouse model of DCM at earlier and later stages of disease progression. Alterations in miRNA and mRNA expression within the heart following hUC-MSC administration were also studied.

Ethics statement

All animal experiments were conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and approved by the Ethics Committee of Hebei Medical University (No.IACUC-Hebmu-2021035).

Experimental Animals

Male C57BL/6J mice of 6-7-weeks age were purchased from Beijing HFK Bio-Technology Co., Ltd. and were housed in a specific pathogen free (SPF) animal facility at Hebei Medical University Laboratory Animal Center. All animals were housed at constant temperature (20 ± 2^oC) and humidity (45–55%), with a 12 hours light/12 dark cycle and free diet and water ad libitum.

Establishment of mouse model of DM and hUC-MSC administration: The groups of experimental animals described here are also the subject of a separate manuscript describing the effects of hUC-MSCs on diabetic nephropathy (He et al., currently under review). A STZ model of DM with low toxicity was established as previously described [53] by intraperitoneal (i.p.) injection of STZ (Cayman Chemicals, catalog no. 18883664) 80 mg/kg body weight in citrate buffer pH 4.5 daily for 5 consecutive days. For each experiment, a control group of non-diabetic (Non-DM) mice was generated by 5 consecutive i.p. injections of citrate buffer alone. Blood glucose concentrations were measured once a week in all mice using a glucometer (Roche, Accu-Chek) and blood glucose test strips (Roche, Excellence). Mice with fasting blood glucose (FBG) levels ≥ 16.7 mM were considered to have DM. Two in vivo experiments were performed of 10 weeks and 18 weeks duration following induction of DM. For each, mice were divided into three groups: Non-DM group, DM group (treated with i.v. injection of sterile saline 2 weeks before termination of the experiment) and DM + MSC group (treated with i.v. injection of hUC-MSCs in sterile saline 2 weeks before termination of the experiment). The experimental designs are illustrated in Fig. 1A. For i.v. treatments, 5 x 10⁵ freshly cultured hUC-MSCs suspended in 0.2 ml of sterile saline or 0.2 ml of sterile saline alone were injected via the tail vein into mice with confirmed DM. Mice were monitored weekly for fasting blood glucose and body weight. An Animal General Distress Scoring Sheet was completed on a daily basis with humane interventions undertaken as appropriate (see Table S1 at the end of the manuscript).

Culture, Identification and Characterization of hUC-MSCs

Cryopreserved hUC-MSCs were purchased from Qilu Cell Therapy Engineering Technology Co., Ltd (Shandong, China). Upon receipt, the hUC-MSCs were thawed, transferred to complete medium (DMEM, low glucose medium with 15% FBS,100 units/ml penicillin, and 100 mg/ml streptomycin) and cultured in a humidified 37°C, 5% CO₂ incubator. The medium was changed every 3 days. When the hUC-MSCs reached 80–90% confluence, the medium was discarded, the cell culture flask was washed three times with PBS, and the cells were lifted with MSC digestion solution (Jing Meng, Beijing, China).

To confirm the expected surface marker phenotype, freshly-lifted hUC-MSCs were stained with fluorochrome-labelled mouse anti-human antibodies against CD73, CD44, CD29, CD105, CD90, CD45, and HLA-DR and with appropriate mouse isotype control antibodies using the BD Biosciences Human MSC Analysis Kit (catalog no. 562245, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Flow cytometry was performed on a BD FACS Calibur (BD Biosciences) and the resulting data files were analyzed using FlowJo Software (Treestar, Ashland, OR).

To confirm tri-lineage differentiation capacity, hUC-MSCs were cultured in 6-well plates in adipogenic differentiation (Cyagen Biosciences, Guangzhou, China, catalog no. HUXUC-90031) or osteogenic differentiation media (Cyagen Biosciences, Guangzhou, China, catalog no. HUXUC-90021). For chondrogenic differentiation, hUC-MSCs were seeded at passage 4 in 15 ml sterile centrifuge tubes in chondrogenic differentiation medium (Cyagen Biosciences, Guangzhou, China, catalog no. HUXUC-90041). The induction of differentiation was confirmed by standard protocols from the manufacturer for adipogenesis (Oil red O staining), osteogenesis (Alizarin red staining) and chondrogenesis (Alcian blue staining) compared to hUC-MSCs cultured under baseline conditions at the same passage number served as negative controls.

Echocardiography: Two-dimensional, M-mode echocardiogram and tissue Doppler were performed using a Vevo 2100 ultrasound system under anesthesia with isoflurane at one day before euthanasia. In order to comprehensively evaluate the systolic and diastolic functions of the heart, the parasternal short-axis scan and the apical four-chamber scan were selected for assessment. Using the parasternal short-axis scan, the following parameters were recorded: heart rate (HR), left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), left ventricular anterior wall thickness at end-diastole (LVAWd), left ventricular anterior wall thickness at end-systole (LVAWs), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs), left ventricular weight (LVW), stroke volume (SV), cardiac output (CO), left ventricular ejection fraction (LVEF) and fractional shortening (FS). The first six indicators measured by ultrasound images can reflect the shape of the heart at the end of systole or diastole. The remaining indicators were calculated based on the first six indicators. LVEF and FS are the most typical indicators to evaluate cardiac systolic function [54]. By the apical four-chamber scan, we measured the ratio of peak early diastolic filling velocity to late atrial filling velocity (E/A) and isovolumic relaxation time (IVRT) which mainly reflect the diastolic function of the heart [55].

Tissue harvest, histopathology staining and image analysis

Following humane euthanasia, mouse hearts were isolated, weighed, fixed in paraformaldehyde and then embedded in paraffin. For each paraffin-embedded heart, whole-organ 4-µm thick sections were prepared using a microtome. For initial assessment of tissue quality and structure, a single heart section from one animal per group was stained with hematoxylin and eosin (H&E) and inspected by light microscopy. Next, for each experiment, whole heart sections from 3 different levels of the organ were stained with Masson’s trichrome (MT) for 6 mice per group. The protocols used for H&E and MT staining are provided in Supplemental Methods. For image analysis of MT-stained sections, 6 fields from each section were selected for the interstitial area, and 3 fields were selected for the perivascular area. Images were collected at 200×magnification and the MT-stained sections were analyzed in blinded fashion using a light microscope (Olympus BX53, Japan). Quantitative image analysis was performed using Image-Pro Plus 6.0 software (Image-pro Plus, Media Cybernetics, Inc., USA). For quantification of interstitial fibrosis, collagen volume fraction (CVF) was calculated for each field as the ratio of the blue-stained area to the total interstitial area. For quantification of perivascular fibrosis, the ratio of the blue-stained perivascular collagen area to the total luminal area (PVCA/LA) was calculated for each vessel within the field [56]. In total 90 fields per heart were analyzed for interstitial fibrosis and 45 fields per heart were analyzed for perivascular fibrosis. The final CVF and PVCA/LA values, expressed as %, were derived for each heart from the average values of all analyzed fields.

Selection of candidate differentially expressed miRNAs and mRNAs from publically available sources: In order to identify miRNAs with altered expression in diabetic mouse heart, we selected Non-DM and DM group sequencing data from a miRNA high-throughput sequencing (series GSE210036 [15]) and generated a heatmap of the top 50 differentially-expressed miRNAs. From this, we selected six fibrosis-associated miRNAs: let-7f, miR-26a, miR-29a, miR-29b, miR-29c and miR-133a. Based on the recent review by Z-Q Jin [57] we also selected three additional candidate miRNAs: miR-34a, miR-155 and miR-326. The relative expression of these nine miRNAs, and subsequently, the relative expression of candidate mRNAs in heart tissue from the experimental groups of the current study were then determined by qRT-PCR (methods described below).

To identify candidate mRNAs with altered expression in the heart in diabetes, we also selected publically available Non-DM and DM group data from a recently performed mRNA high-throughput sequencing analysis (series GSE161052 [17]) and generated a volcano plot with annotation of the top 18 differentially expressed mRNAs. We noted that these mRNAs included transcripts encoding the alpha 1 chains of collagen I and collagen III and selected these mRNAs for qRT-PCR analysis in hearts from the current study due to their relevance to fibrosis and prior identification of COL1A1 as a target for miRNA-133a. In addition, 6 other fibrosis-related mRNAs potentially regulated by miRNA-133a - α-SMA, TGF-β, Smad2, Smad3, Smad4 and FGF1 were also selected for quantitative analysis in heart tissue from our experimental groups.

RNA extractions and quantitative reverse transcription and polymerase chain reaction (qRT-PCR) analyses

After euthanasia, one-half of the fresh heart tissue was stored in a -80 ° C freezer for further use (n = 6 per group). For qRT-PCR analyses, total miRNA or mRNA were extracted from heart tissue using miRNA extraction and isolation kit (DP501, TIANGEN, Beijing) and mRNA extraction and isolation kit (DP424, TIANGEN, Beijing) respectively according to the manufacturers’ instructions. The miRNA and mRNA preparations were transcribed to cDNA by miRNA First Strand cDNA Synthesis (Tailing Reaction, B532451, Sango Biotech, Shanghai) and cDNA first strand synthesis kit (ZS-M14003, ZHONGSHI TONTAU, Tianjin), respectively according to the manufacturers’ instructions. Quantitative PCR was performed with a SYBR Green PCR master mix (ZS-M13010, ZHONGSHI TONTAU, Tianjin). Because miRNA was generally composed of more than 20 bases, different from general mRNA, we adopted the poly(A) tailing method for reverse transcription of miRNA. The tailed reverse transcription assay utilizes poly (A) polymerase for mature miRNA plus poly (A) tail, followed by 5 '- end universal tagged oligo dt as the reverse transcription primer to obtain the human elongated miRNA cDNA first strand, and finally fluorescence quantitative PCR detection with a reverse primer complementary to the universal tag sequence by dye method or probe method. Universal PCR primer R (B661601-0002, Sango Biotech, Shanghai) were the same for all miRNAs and internal control U6. The primer F sequences of miRNA are listed in Table S2 and primer sequences of mRNA are listed in Supplemental Table S3. A melt curve was included to ensure primer specificity. Experiments were performed in triplicate, and results were normalized to U6 or β-actin expression (2^−ΔΔCT method).

Statistical analysis

For all experiments, group data were expressed as mean ± SD and analyzed using GraphPad Prism 6. The differences between the groups were compared using ordinary one-way analysis of variance (ANOVA) or multiple comparisons of 2-way ANOVA. P < 0.05 was considered statistically significant. For bioinformatics analyses of publically available miRNA and mRNA datasets, R programming language (version 4.1.3) and R studio (version 3.3.0) were used.

Characterization of human umbilical cord-derived mesenchymal stromal cells

For validation of hUC-MSC cultures used for the in vivo studies, it was first determined whether the cells met the International Society for Cellular Therapy (ISCT) minimal criteria. The cells demonstrated an adherent growth pattern, were highly proliferative within serum-containing medium (data not shown), exhibited multi-lineage differentiation in assays of adipogenesis, osteogenesis and chondrogenesis (Fig. 1A-C), and demonstrated the expected positive and negative staining ratios of related surface markers in flow cytometry analyses [CD29 (98.91%), CD44 (99.43%), CD73 (96.78%), CD90 (98.22%), CD105 (98.56%), CD45 (0.88%), HLA-DR (0.5%)] (Fig. 1D).

Establishment of mouse model of diabetes mellitus with systemic hUC-MSC administration

Groups of mice with DM, along with Non-DM control groups, were established by intraperitoneal injection of streptozotocin (STZ) or citrate buffer respectively and were used for two, 3-group in vivo experiments involving i.v. injection of hUC-MSCs or vehicle (sterile saline) at earlier (8 weeks) and later (16 weeks) time-points followed by 2 week observation periods before euthanasia (Fig. 2A, B). As shown in Fig. 2C-F, the fasting blood glucose (FBG) of both groups of DM mice increased significantly while the body weights decreased throughout the time courses of the two experiments compared to the Non-DM groups. Notably, for both experiments, there were no differences between the DM and DM + MSC groups in FBG or body weight (Fig. 2C-F). These results confirmed establishment of a model of DM for up to 18 weeks and indicated that i.v. administration of single doses of hUC-MSCs at 8 and 16 weeks following induction of DM did not ameliorate hyperglycemia or weight loss.

Effects of early or later hUC-MSC administration on heart weights of diabetic animals

At the 10 week time-point (following hUC-MSC injection at 8 weeks), the heart weights of mice in the DM group and the DM + MSC groups were significantly less than those of the Non-DM group (Fig. 2G), while heart-to-body weight ratio did not differ among the three groups (Fig. 2F). For this earlier time of administration, no difference in heart weight was seen between DM and DM + MSC groups. At the 18 week time-point (following hUC-MSC injection at 16 weeks), the heart weight of mice in the DM group was not different to those of the Non-DM group and the heart-to-body weight ratio was higher (Fig. 2G, H). In contrast, for this later time-point of hUC-MSC injection, the heart weights of the DM + MSC group remained significantly lower than those of the Non-DM group and the heart-to-body weight remained similar (Fig. 2G, H). Comparing results across the two time-points for the DM group, both heart weight and heart-to-body weight ratio increased substantially between 10 and 18 weeks and these trends were largely prevented by hUC-MSC administration (Fig. 2G, H). Thus, while early administration of hUC-MSCs did not prevent the loss of heart weight following 10 weeks of DM, the progressive increase in heart weight and heart-to-body-weight ratio that occurred between 10 and 18 weeks after DM onset was prevented by a later injection of hUC-MSCs.

Effects of early or later hUC-MSC administration on heart function of diabetic animals

Echocardiography was used to evaluate the effects of DM and i.v. hUC-MSC administration on cardiac function. Measurements were taken via the parasternal short-axis and the apical four-chamber views (Fig. 3A). Among the measured indicators, left ventricular ejection fraction (LVEF) and fractional shortening (FS) reflected systolic function (Fig. 3B, C), while E/A ratio and isovolumetric relaxation time (IVRT) mainly reflected diastolic function (Fig. 3D, E). As shown, in comparison to the Non-DM group, mice in the DM group developed cardiac systolic (reduced EF and FS) and diastolic (reduced E/A ratio and ICRT) dysfunction as early as 10 weeks after STZ injection. The systolic dysfunction worsened between 10 and 18 weeks. For both time-points, hUC-MSC administration was associated with significant improvements in systolic and diastolic function compared to control DM groups. Results for other echocardiographic parameters are summarized in Table 1. The echocardiographic studies provided evidence for progressive cardiac dysfunction in the mouse model of DM and for beneficial effects of single i.v. injections of hUC-MSCs following both shorter and longer DM duration.

Table 1

Summary of results of cardiac functional indices from echocardiography of three groups of mice each from experiments of 10 weeks and 18 weeks duration
	Non-DM group	DM group	DM + MSC group
Heart Rate(bpm)
10 wks	501.6 ± 44.3	380.6 ± 50.4 *	389.3 ± 44.4 *
18 wks	515.7 ± 47.1	375.8 ± 55.8 *	382.6 ± 66 *
LVIDd(mm)
10 wks	3.6 ± 0.4	4.0 ± 0.2 *	3.6 ± 0.3
18 wks	3.7 ± 0.3	4.1 ± 0.4 *	3.6 ± 0.2 #
LVIDs(mm)
10 wks	2.7 ± 0.3	3.2 ± 0.3 *	2.6 ± 0.3 #
18 wks	2.6 ± 0.3	3.2 ± 0.4 *	2.6 ± 0.4
SV(µL)
10 wks	40.1 ± 4.4	30.9 ± 1.9 *	36.2 ± 2.8 *#
18 wks	39.4 ± 5.5	29.4 ± 2 *	35 ± 3.4 *
CO(mL/min)
10 wks	19.8 ± 2.2	13.7 ± 3.1 *	14.4 ± 2.9 *
18 wks	20.2 ± 2.7	12.5 ± 1.4 *	14 ± 2.8 *
LVAWd(mm)
10 wks	0.9 ± 0.1	0.7 ± 0.1 *	0.7 ± 0.1 *
18 wks	0.9 ± 0.1	0.8 ± 0.1	0.7 ± 0.1 *
LVAWs(mm)
10 wks	1.3 ± 0.1	1 ± 0.1 *	1 ± 0.1 *
18 wks	1.4 ± 0.1	1.1 ± 0.1 *	1 ± 0.1 *
LVPWd(mm)
10 wks	0.8 ± 0.1	0.7 ± 0.2	0.7 ± 0.1
18 wks	0.8 ± 0.1	0.7 ± 0.1	0.7 ± 0.1
LVPWs(mm)
10 wks	1.2 ± 0.1	1 ± 0.1 *	1 ± 0.1 *
18 wks	1.2 ± 0.1	1 ± 0.1 *	1 ± 0.1 *
LVW(mg)
10 wks	84.7 ± 7.4	67.1 ± 8.2 *	66.9 ± 5.8 *
18 wks	93.5 ± 8.5	76.6 ± 5 *	70.4 ± 10 *#
Abbreviations: Non-DM = non-diabetic group; DM = diabetes mellitus with injection of saline; DM + MSC = diabetes mellitus with injection of hUC-MSCs; HR = heart rate; LVIDd = left ventricular internal diameter at end-diastole; LVIDs = left ventricular internal diameter at end-systole; SV = stroke volume; CO = cardiac output; LVAWd = left ventricular anterior wall thickness at end-diastole; LVAWs = left ventricular anterior wall thickness at end-systole; LVPWd = left ventricular posterior wall thickness at end-diastole; LVPWs = left ventricular posterior wall thickness at end-systole; LVW = left ventricular weight. All values are expressed as means ± SD. n = 6 per group. * = significant (p < 0.05) compared to Non-DM group, # = significant (p < 0.05) compared to DM group. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.

Effects of early or later hUC-MSC administration on myocardial structure of diabetic animals

H&E staining of cardiac tissue sections revealed some disorganized patterning of cardiomyocytes in the left ventricles of DM and DM + MSC groups at both time-points compared to Non-DM hearts (Fig S1.). For more quantitative assessment of myocardial structure, the extent of interstitial and perivascular fibrosis was compared among the groups by Masson’s trichrome staining and image analysis of hearts collected at 10 weeks (Fig. 4A, C) and 18 weeks (Fig. 4B, D) after induction of DM. As shown in Figs. 4C and D, DM was associated with significantly increased % collagen vascular fraction (CVF) in the myocardial interstitium and increased % perivascular collagen area/luminal area (PVCA/LA) compared to Non-DM. Perivascular fibrosis increased in all 3 groups between 10 weeks and 18 weeks but the increase was more marked in the DM group. For both earlier and later time-points, hUC-MSC administration resulted in prevention of the DM-associated intra-cardiac fibrotic changes.

Effects of early or later hUC-MSC administration on cardiac expression of miRNA and mRNA in diabetic animals

To identify potential candidates for DM-associated, myocardial miRNAs that may be modulated by hUC-MSC administration, we first generated a heatmap (Fig. 5A) of the top 50 differentially expressed miRNAs in publically available datasets for cardiac tissue of groups of male C57BL/6 mice with and without STZ-induced DM for 12 weeks from the recently-published study of Cagnin et al. (GSE210036, [15]). From these, 6 miRNAs were selected for quantitative (q)RT-PCR analysis in heart tissue samples from our two in vivo experiments. An additional 3 miRNAs, selected from the recent review of Z-Q Jin were also quantified [16]. The results of qRT-PCR analysis for these 9 candidate miRNAs are shown in Fig. 5B, C and Fig S2. As shown in Fig. 5B, C myocardial expression of miRNA-133a, an anti-fibrotic miRNA, was reduced in DM compared to Non-DM mice at 10 weeks and 18 weeks but was unchanged in DM-MSC groups at both time-points. For the other eight miRNAs that were studied, none were significantly different between DM and Non-DM groups at either time point (Fig S2).

To screen for mRNAs that have been found to be upregulated within the heart in DM and may be regulated by miRNA-133a, we generated a volcano plot (Fig. 5D) of the differentially expressed transcripts (DM vs. Non-DM) from the publically-available high-throughput mRNA sequencing dataset of Wei et al. (GSE161052 [17]). Among the upregulated transcripts in this analysis, Col1A1 has been shown by Castoldi et al. to be a direct target of miRNA-133a [18]. According to the relevant information in the Targetscan database, we found the possible binding sites of miRNA-133a and Col1A1 (Fig. 5E). We proceeded to perform qRT-PCR of heart tissue samples from our two in vivo experiments to quantify mRNAs for Col1A1 and Col3A1 (Fig. 5F, G). These analyses showed that the two collagen gene transcripts were increased in hearts of DM compared to Non-DM mice at 10 and 18 weeks and that hUC-MSC administration at both earlier and later time-points had prevented the upregulation.

Next, the mRNA expression of other pro-fibrotic gene products - ACTA2(α-SMA), TGFB1(TGF-β), Smad2, Smad3, Smad4 - and the anti-fibrotic mRNA FGF1 was determined in the 10 week and 18 week cardiac tissue samples by qRT-PCR (Fig. 5I, J). As shown, myocardial expression of mRNAs encoding ACTA2, Smad3, and Smad4, was increased in DM compared to Non-DM animals at both time-points. In all cases, hUC-MSC administration was associated with reversal of the change. Strikingly, myocardial expression of mRNA for FGF-1, which has been reported to inhibit fibroblast activation, collagen production and pro-fibrotic TGF-β1 signaling [19], was increased in DM + MSC groups compared to both DM and Non-DM groups (Fig. 5I, J).

It was concluded that the beneficial effects on myocardial function and structure of single i.v. injections of hUC-MSCs at earlier and later time-points following induction of DM are accompanied by preservation of the expression of the anti-fibrotic miRNA, miR-133a, prevention of the up-regulation of pro-fibrotic mRNAs and increased expression of the anti-fibrotic factor FGF-1.

Diabetic cardiomyopathy is a serious complication of diabetes mellitus, which leads to heart failure and increased mortality [20]. Cardiac structural abnormalities are the main pathological changes in DCM, including cardiac fibrosis and cardiac hypertrophy, and these pathological alterations contribute to the development of heart failure symptoms [21–23]. The current clinical treatment of DCM focuses predominantly on relieving these symptoms and, while there has been much progress in pharmacological approaches to the treatment of heart failure, there is no effective method to prevent the progression of myocardial fibrosis in DCM [24]. Due to their paracrine properties and readily accessible source material, hUC-MSCs have been identified as a promising, novel treatment for DCM [25]. Potential mechanisms for their therapeutic benefits include paracrine-mediated immunomodulatory, pro-angiogenic and anti-inflammatory effects [26]. It is worth noting that MSCs have been reported to exhibit both antidiabetic properties and cardioprotective properties, which, in combination, could be highly beneficial for the prevention or reversal of DCM [27]. In keeping with this, intravenously delivered MSCs have been demonstrated to alleviate cardiac inflammation in rodent models of diabetes [28].

In this study we used tail vein injection of hUC-MSCs to observe whether the cardiac dysfunction and pathological changes of fibrosis seen in diabetic mice would be improved. At the same time, the expression changes of mRNA and miRNA were analyzed by RT-qPCR in order to explore the therapeutic target and potential mechanism of action of hUC-MSC administration. To gain more insight into the influence of diabetes duration on the effects of systemic hUC-MSCs on DM and DCM, we performed experiments in which the intervention was made at earlier and later stages of disease. Of interest, even at the earlier time-point, we observed no evidence of an anti-diabetic effect of i.v. hUC-MSCs – likely reflecting the fact that DM due to islet destruction by STZ had been established for 8 weeks or more before cells were administered. For the purpose of interpreting our results, this indicates that the observed improvements in cardiac function and structure following hUC-MSC infusion likely reflect direct effects on DCM pathophysiology rather than indirect benefits due to reduction in hyperglycemia. Our application of echocardiography provided a comprehensive non-invasive evaluation of heart and function and has been used by others to demonstrate beneficial effects of therapeutic interventions in models of DCM [29]. It also provides a means to compare cardiac functional abnormalities observed in the mouse model, with those reported for human subjects with DCM, in whom diastolic dysfunction (reflected by E/A and IVRT) may be more prevalent and occur earlier then systolic dysfunction [30]. In keeping with this, there was marked increase in IVRT by 10 weeks after the induction of DM in our experiments which persisted at 18 weeks and was largely prevented by hUC-MSC injection. Nonetheless, evidence of systolic dysfunction and of its amelioration by hUC-MSCs was also observed by echocardiography at both time-points. Consistent with these functional abnormalities, our quantitative microscopic analysis of Masson’s trichrome-stain cardiac tissue from the same animals, revealed significant DM-associated increases in interstitial and perivascular fibrosis which were reduced in recipients of hUC-MSCs. Taken together, the functional and structural abnormalities observed in our DM groups are concordant with previous preclinical studies in which significant cardiac dysfunction and myocardial fibrosis were observed in STZ-induced models of DM of 8 weeks [31] and 14 weeks [32] duration. Strikingly, our results indicate that single i.v. injections of hUC-MSCs are capable of potently modulating the underlying pathophysiology of DCM at varying stages of its progression toward severe cardiac dysfunction.

Umbilical cord-derived MSCs are an important member of the growing family of MSC-based therapeutic products and are increasingly gaining attention. Their readily accessible source and their capacity for promotion of tissue repair and immune regulation make them particularly well suited to treating diseases involving tissue degeneration caused by chronic inflammation [33] [34] [35]. Based on these properties, hUC-MSCs have been tested in pre-clinical and clinical studies involving a range chronic disease states including arthritis, stroke, liver disease, diabetes mellitus, systemic lupus erythematosus and cardiovascular disease [36]. In the case of DCM, however, previous preclinical experimental studies of MSCs mainly involved the administration of bone marrow (BM)- and adipose tissue (AT)-derived MSCs [32, 37]. These preclinical experimental studies, as well as one involving the administration of Wharton's jelly derived-MSCs [38], did not compare the disease progression and effects of MSC administration at both early and later stage of DCM.

Cardiac fibrosis is one of the most common pathological changes in DCM, which eventually leads to cardiac dysfunction and heart failure [39]. In DCM, myocardial fibrosis is a chronic and progressive process characterized by the accumulation of collagen fibers in the myocardium in the setting of prolonged hyperglycemia [40, 41]. Jin et al. demonstrated that adipose-derived MSC administration was beneficial in a rat models of DCM when administered 8 weeks after STZ. [42]. We confirmed this finding with hUC-MSCs and extended to observation of benefit to a later time of intervention. Although both interstitial fibrosis was increased by DM at both time-points and was ameliorated by a single, relatively late i.v. injection of hUC-MSCs, it was notable that only peri-vascular fibrosis had worsened significantly between 10 and 18 weeks in DM mice and that this accelerated advancement was effectively prevented by hUC-MSCs

MicroRNAs are known to be key modulators of the post-transcriptional regulation of genes in all cells types [43]. Current research confirms that miRNAs play important roles in pathological processes in the cardiovascular system, including arrhythmia, cardiac hypertrophy, cardiac fibrosis and myocardial infarction [44]. For this reason, miRNAs have emerged as novel targets for DCM treatment and important tools for disease diagnosis, prevention, and treatment [45]. It is known that MSCs release large amounts of extracellular vesicles (EVs) containing various bioactive molecular including a range of miRNAs and may, therefore, be a potent sources of transferable miRNAs in disease settings [46]. Based on the sequencing results in the literature, we selected a panel of pro-fibrotic miRNAs such as let-7f, miRNA-26a, miRNA-29a, miRNA-29b, miRNA-29c, miRNA-34, miRNA-133a and some anti-fibrotic miRNAs such as miRNA-155, miRNA-326 to assess the expression levels of fibrosis-related miRNAs by RT-qPCR. This revealed that the expression of miRNA-133a in the hearts of diabetic mice was significantly lower than that in normal mice and that this reduction was attenuated in the groups that received hUC-MSCs at both time points assessed. It has previously been shown that cardiac miR-133a is down-regulated in DM and that miR-133a overexpression in the heart (through cloning the genomic sequence of miR-133a into the alpha-myosin heavy-chain promoter) prevented early cardiac fibrosis in DM mice [47]. Our study is the first, to our knowledge, to demonstrate that miRNA − 133a dysregulation can be reversed by hUC-MSC delivery with consequent reduction in fibrosis.

The regulation of target genes (mRNAs) by miRNAs occurs at the post-transcriptional level, mainly through the inhibition of their translation to protein. There are many studies confirming that miRNA-133a regulates fibrosis in various organs such as heart, lung, and kidney through downstream mRNAs [47, 48]. Thus, we also examined the intra-cardiac expression of mRNAs encoding the collagen chains COL1A1 and COL3A1 (the former being a known target of miRNA-133a), along with mRNAs encoding other pro-fibrotic proteins, ACTA2(α-SMA), TGFB1(TGF-β), Smad2, Smad3, Smad4, and the anti-fibrotic mediator FGF1. Collagen I and collagen III are the main components of extracellular matrix (ECM), which is central to wound healing and fibrosis [49]. Our results are consistent with the potential role of miR-133a downregulation to enhance the expression of COL1A1(Collagen I) and with the reversal of this pro-fibrotic mechanism by systemic hUC-MSC therapy. α-SMA(ACTA2) is one of six different actin isoforms involved in smooth muscle contraction. It is considered to be a marker of myofibroblasts, and its gene expression level correlates with the degree of tissue fibrosis [50]. Similarly, the TGF-β/Smad (Smad2, Smad3, Smad4) signaling pathway is an important driver of fibrocyte division and collagen deposition and is an recognized target for regulating fibrosis [51]. In addition to its modulatory effects on fibrosis, FGF1 has also been reported to be a potent insulin sensitizer [52]. Taken together, the results for these mRNA quantification studies were in keeping with a potent anti-fibrotic effect of both earlier and later hUC-MSC injection, resulting in amelioration of the degree of interstitial and perivascular fibrosis and improved cardiac systolic and diastolic function.

The results we present here have demonstrated that single i.v. injections of hUC-MSCs improved cardiac function and myocardial fibrosis in mice with diabetes of shorter and longer duration. These findings extend the body of evidence to support the clinical use of hUC-MSCs in for prevention or treatment of symptomatic DCM. Mechanistically, our findings are consistent with a mode of action whereby systemically administered hUC-MSCs either directly or indirectly reverse diabetes-associated suppression of the anti-fibrotic miRNA, miR-133a within the myocardium.

AD-MSCs

Adipose-Derived Mesenchymal Stem Cells

AGS

Animal General Scoring

ANOVA

Ordinary one-way analysis of variance

BM-MSCs

Bone Marrow Mesenchymal Stem Cells

Cardiac output

CVF

Collagen volume fraction

DCM

Diabetic cardiomyopathies

Diabetes mellitus

E/A

The ratio of peak early diastolic filling velocity to late atrial filling velocity

EVs

Extracellular vesicles

FBG

Fasting blood glucose

Short axis fractional shortening

H&E

Hematoxylin and eosin

Heart failure

Heart rate

hUC-MSCs

Human umbilical cord mesenchymal stromal cells

ISCT

International Society for Cellular Therapy

IVRT

Isovolumic relaxation time

LVAWd

Left ventricular anterior wall thickness at end-diastole

LVAWs

Left ventricular anterior wall thickness at end-systole

LVEF

Left ventricular ejection fraction

LVIDd༚ Left ventricular internal diameter at end-diastole

LVIDs

Left ventricular internal diameter at end-systole

LVPWd

Left ventricular posterior wall thickness at end-diastole

LVPWs

Left ventricular posterior wall thickness at end-systole

LVW

Left ventricular weight

miRNA

microRNA

PCR

Polymerase chain reaction

PVCA/LA

The ratio of perivascular collagen area to luminal area

SPF

Specific pathogen free

STZ

Streptozotocin

Stroke volume

WJ-MSCs

Wharton Jelly Cells

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

MD Griffin reports honoraria from the American Society of Nephrology; Théa Pharma Ltd., Ireland; and Novo Nordisk; research funding from Orbsen Therapeutics Ltd.; and 3

advisory roles as an Editorial Board member for the journals Transplantation and Frontiers in Pharmacology and an Associate Editor for Mayo Clinic Proceedings.

The other authors declare that they have no competing interests.

Funding

This work was supported by: Natural Science Foundation of China (Grant No. 81801278), Natural Science Foundation of Hebei Province (Grant No. H2019206637), China Scholarship Council (Grant No. 201608130015), Hebei University Science and technology research project (Grant No. ZD2019049), Excellent Overseas researcher Program in Hebei Provincial Department of Human Resources and Social Security (Grant No. C20190509), and Key Natural Science Foundation of Hebei Province (H2020206557). MDG and TO’B are also supported by grants from the European Commission [Horizon 2020 Collaborative Health Project NEPHSTROM (grant number 634086; TO’B, MDG)] and Science Foundation Ireland [CÚRAM Research Centre (grant number 13/RC/2073_P2; TO’B, MDG) and Investigator Programme (grant number 15/IA/3136; TO’B)] and by the European Regional Development Fund. The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript

Authors' contributions

LBX participated in conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing. HJJ, YW and FBF participated in conception and design, data analysis and interpretation. GRY, DXF and LX participated in data analysis and interpretation. MDG, SOH, SS and YL participated in conception and design, reviewed the manuscript. TOB, JM and HC participated in conception and design, reviewed the manuscript, and gave final approval for manuscript submission.

Acknowledgements

Not applicable.

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Human umbilical cord-derived mesenchymal stromal cells improve myocardial fibrosis and restore miRNA-133a expression in diabetic cardiomyopathy

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