| Diabetes is a global health care issue.The prevalence of diabetes has steadily increased in many parts of the world and is expected to continue increasing as obesity and other clinical risk factors for diabetes become more common.The vascular complications of diabetes are among the most serious manifestations of the disease.Diabetes Mellitus is a major risk factor of peripheral artery disease,leading to increased morbidity and mortality as well as an accelerated disease course.Critical limb ischemia(CLI)is an advanced stage of peripheral artery disease.Diabetes increases approximately twofold to fourfold the incidence and severity of critical limb ischemia.Diabetes is also an independent risk factor for postoperative amputation and complications in CLI.CLI is often the result of the blockage of the arteries that supply blood to lower limbs,setting off a cascade of pathophysiologic events that ultimately lead to rest pain or trophic lesions of the legs.The first response of our body against ischemia is to induce angiogenesis;however,this endogenous potential is disrupted in hyperglycemic condition due to the dysfunctions of cells involved in angiogenesis,such as endogenous cells,smooth muscle cells and macrophage.Such vascular abnormalities that cause CLI in diabetes patients severity increases with worsening blood glucose and duration of diabetes.Current recommendations suggest arterial reconstruction by using surgical intervention,who have a predicted 1-year amputation-free survival of at least 75%,as the first choice for the treatment of CLI.However,many patients,especially patients with large surface of injury and with high re-occurrence such as those with diabetes mellitus,have been assumed with "no choice" for surgical intervention.Therapeutic angiogenesis which aims to induce the generation of new blood vessels,had been assumed as the most potential treatment for CLI.Because CLI is characterized by chronically hypoxic conditions in muscle and epithelial tissues for weeks or months,the promotion of regional angiogenesis can produce clinical benefit,including the relief of rest pain,healing of ischemic ulcers,and a reduction in the incidence of major limb amputation.During the past 2 decades,attempts had been made to induce therapeutic angiogenesis by using recombinant proteins,genetic approach,and stem cells.However,there is no effective drug-based treatment for CLI in diabetes at present.Skeletal muscle is the largest secretory organ which could secrete numbers of paracrine and autocrine signals including angiogenic factors.Our previous studies showed that skeletal muscle cells could simultaneously secrete a number of paracrine signals including angiogenic factors,which in turn could enhance cell-cell communication between skeletal muscle cells and cells involved in angiogenesis,such as endothelial and smooth muscle cells.These paracrine signals affect the biological functions of cells involved in angiogenesis,and subsequently,promote neovascularization.However,hyperglycemia disrupts the secretory function of skeletal muscle cells,and thus,upregulating this biological function of skeletal muscle cells has become an obstacle for inducing neovascularization in diabetes patients.Over the past few decades,Traditional Chinese Medicine has been confirmed to be effective and promising in the therapy of diabetes and its complications with few undesirable effects compared with the modern drugs.In this study,we use salidroside to promote neovascularization in diabetic condition.Salidroside is the main compound of Rhodiola,a medicinal plant found in mountains at high altitudes.Rhodiola itself has been used in traditional medicine for a long time,owing to its adaptogenic and antifatigue properties.Salidroside displays a broad spectrum of pharmacological properties,such as resist anoxia,anti-fatigue,anti-oxidation,and enhance cell survival.However,whether or not salidroside could induce neovascularization in diabetic condition remains unknown.We found that in hyperglycemic condition,the expression levels of angiogenic factors such as vascular endothelial growth factor(VEGF)and platelet-derived growth factor(PDGF)-BB in skeletal muscle cells significantly decrease.Various studies identified that expression of VEGF and PDGF-BB are critically important in the formation of collateral vessels in response to ischemia.On the other hand,hyperglycemia induced the expression of prolyl hydroxylase domain 3(PHD3),a member of the prolyl hydroxylase domain family which had been known to enhance the degradation of hypoxia-inducible factor(HIF)-1?(11)a master regulator of various angiogenic factors,in skeletal muscle cells.Silencing of skeletal muscle cells PHD3 restored the expressions of VEGF and PDGF-BB suppressed by hyperglycemic condition.Thus,these results indicated that upregulation of PHD3 might be the reason for the downregulation of angiogenic factors in hyperglycemia.We also found that addition of salidroside cancelled the upregulation of PHD3 by hyperglycemia,and concomitantly,upregulated VEGF and PDGF-BB expressions in skeletal muscle cells.Furthermore,salidroside could also restore the proliferation and migration potential of skeletal muscle cells suppressed by hyperglycemia.VEGF and PDGF-BB are crucial angiogenic factors that affects the proliferation and migration of both endothelial and smooth muscle cells.Concomitant with the results that the expression levels of these factors in skeletal muscle cells were suppressed in hyperglycemia,the mobility and proliferation potentials of endothelial and smooth muscle cells in conditioned medium obtained from skeletal muscle cells cultured in hyperglycemic condition were also disrupted.However,when conditioned medium obtained from salidroside-treated skeletal muscle cells cultured in hyperglycemic condition was used,these potentials were restored,indicating that salidroside-induced skeletal muscle cell paracrine signaling is sufficient to affect the biological functions of endothelial and smooth muscle cells in hyperglycemic condition.Furthermore,our results showed that overexpression of PHD3 decreased the effect of salidroside in inducing the mobility and proliferation of endothelial and smooth muscle cells in high glucose condition,indicating that salidroside exerts its effects on the biological functions of endothelial and smooth muscle cells via inhibiting PHD3.Finally,we investigated whether salidroside could promote neovascularization in ischemic tissue of diabetic mice model.Our results demonstrated that salidroside was effective to promote blood perfusion recovery in ischemic hind limb of streptozotocin-induced diabetic mice,most plausibly by suppressing the expression level of skeletal muscle cells PHD3 induced by hyperglycemia,which in turn promoted the paracrine signaling,especially VEGF and PDGF-BB,of skeletal muscle cells.As the results,treatment with salidroside elevated the migration and proliferation potentials of endothelial and smooth muscle cells repressed by hyperglycemia,and subsequently,enhanced neovascularization and blood perfusion recovery in the ischemic hind limb of diabetic mice.In conclusion,our study showed,for the first time,the efficacy of salidroside in inducing neovascularization in diabetic CLI mice model,most plausibly by enhancing the paracrine function of skeletal muscle cells.Although further studies,including the side effect of salidroside and more detailed pre-clinical studies are needed,our novel findings demonstrate that salidroside might be a potential small molecule drug for therapeutic angiogenesis in treating diabetes CLI. |
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