Diabetes mellitus is a major risk factor for development of myocardial infarction in a large segment of the population, causing an enormous economic burden. A number of studies have shown that diabetic hearts are more sensitive to ischemic insults that can lead to heart failure [25, 26]. Development of diabetic cardiomyopathy is associated with decreased diastolic compliance, increased interstitial fibrosis, and myocyte hypertrophy . Accrual of reactive oxygen species (ROS) in a diabetic heart leads to endothelium dysfunction characterized by vascular remodeling, disappearance of capillary endothelium, and altered gene and protein expression in endothelial cells . Cell-based therapies using MSCs have been successfully used for treatment of the damaged heart, yet stem cells isolated from diabetic animals have severely reduced reparability , resulting in diminished heart repair after autologous transplantation . Therefore, the present study demonstrated that preconditioning with HG/H-CCM can augment survival, proliferation, and angiogenic ability of MSCs isolated from streptozotocin-induced diabetic mice.
Bone marrow-derived MSCs have been shown to be the best candidates for myocardial regeneration and possess the ability to form cardiomyocytes, endothelial cells, and smooth muscle cells [29, 30]. Adoptive transfer of MSCs in a rat model of diabetic cardiomyopathy is associated with enhanced angiogenesis, myogenesis, and cardiac function [31, 32]. Nevertheless, the success of MSCs therapy for diabetic heart repair has been accompanied by reports of functional MSC decline as a consequence of diabetes. MSCs from streptozotocin-induced diabetic rats have impaired abilities for proliferation, paracrine, antiapoptosis, and myogenic differentiation . Similarly, the onset of diabetes can attenuate the osteogenic differentiation ability of MSCs . Therefore, augmentation of depleted MSC function becomes imperative if MSCs are to be used for autologous therapy to repair diabetic heart failure.
Preconditioning of stem cells with growth factors , hypoxic shock , and antiaging  compounds represents an effective strategy to enhance survival, proliferation, and differentiation of MSCs. Recent studies showed that ischemic preconditioning is a powerful regulator of cardioprotection [36–38], and we hypothesized that short-term stimulation of cardiomyocytes with a combination of stress stimuli can mimic in vitro the effects of ischemic preconditioning observed in vivo. Therefore, the normal and diabetic microenvironments of the myocardium were simulated in vitro by treating NRCMs with a combination of oxidative stress and high glucose, with the premise that treatment of dmMSCs with HG/H-CCM can enhance survival, proliferation, and importantly, increase the angiogenic ability of dmMSCs. We showed previously that diabetic mouse-derived MSCs have impaired proliferation, antioxidant levels, and survival signaling , meriting the need for preconditioning. Increased VEGF levels were observed in conditioned medium from NRCMs treated with HG/H and concurs with previous reports showing upregulation of VEGF as a consequence of hypoxia . In parallel to medium preconditioning, dmMSCs were preconditioned with VEGF alone to highlight that HG/H-CCM may contain other angiogenic and nonangiogenic paracrine factors, and these factors collectively can produce the optimal cellular response compared with treatment with VEGF alone.
dmMSCs were preconditioned with HG/H-CCM in parallel with VEGF. HG/H-CCM preconditioning significantly increased survival and proliferation of dmMSCs compared with treatment with VEGF alone and nontreated control. Our study also shows that the HG/H-CCM preconditioning favors commitment of dmMSCs to the endothelial lineage, as confirmed by RT-PCR expression of endothelial lineage markers CD31 and CD34, concomitant with increased tube formation and nitric oxide production. These results concur with previous reports showing that oxidative stress can enhance recruitment , survival [34, 40], migration , and differentiation  of MSCs. Furthermore, it has been shown that oxidative stress can augment the angiogenic potential of age-depleted bone marrow and adipose-derived MSCs  and concurs with our observed results showing increased angiogenic ability of diabetic MSCs after preconditioning with HG/H-CCM.
Adoptive transfer of HG/H-CCM-preconditioned dmMSCs in diabetic animals showed augmented cardiac function and was concomitant with reduced collagen deposition and apoptosis, compared with nontreated dmMSCs and diabetic controls. Interestingly, transplantation of HG/H-CCM-preconditioned dmMSCs resulted in significant augmentation of angiogenesis in the diabetic heart, evidenced by increased SMA+ cells and eNOS expression, whereas iNOS expression was significantly reduced compared with animals transplanted with nontreated dmMSCs and diabetic controls. Gene-expression analysis of hearts from animals transplanted with HG/H-CCM-preconditioned dmMSCs showed increased angiogenic (VEGF, ANG-1) and cardiac (MEF2c, GATA-4, NKx2.5) markers but decreased expression of NF-κB compared with animals with nontreated dmMSCs and diabetic controls. Conversely, increased paracrine factors (IGF-1, FGF-2, SDF-1, HGF) were found in the hearts of animals transplanted with CCM-preconditioned dmMSCs, supporting the idea that preconditioned dmMSCs have better survival, angiogenic ability, and can improve heart function by augmentation of a diabetic environment, possibly through a paracrine-mediated effect.
MSCs represent an attractive option for cell-based therapies for the repair of the diabetic heart. However, MSCs from a diabetic animal have attenuated ability to survive, proliferate, and repair the heart. Therefore, we showed that preconditioning with HG/H-CCM can augment survival, proliferation, and the angiogenic ability of dmMSCs. Furthermore, adoptive transfer of dmMSCs preconditioned with HG/H-CCM improves cardiac function and angiogenesis while reducing fibrosis and apoptosis in the diabetic heart, possibly through a paracrine effect. Thus, preconditioning of dmMSCs represents an effective strategy to improve depleted MSC function and holds promise for the treatment of diabetic heart failure.