Abstract
IntroductionDiabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties. In myoblasts and muscle satellite cells HO-1 improves survival, proliferation and production of proangiogenic growth factors. Induction of HO-1 in injured tissues facilitates neovascularization, the process impaired in diabetes. We aimed to examine whether conditioned media from the HO-1 overexpressing myoblast cell line can improve a blood-flow recovery in ischemic muscles of diabetic mice.MethodsAnalysis of myogenic markers was performed at the mRNA level in primary muscle satellite cells, isolated by a pre-plate technique from diabetic db/db and normoglycemic wild-type mice, and then cultured under growth or differentiation conditions. Hind limb ischemia was performed by femoral artery ligation in db/db mice and blood recovery was monitored by laser Doppler measurements. Mice were treated with a single intramuscular injection of conditioned media harvested from wild-type C2C12 myoblast cell line, C2C12 cells stably transduced with HO-1 cDNA, or with unconditioned media.ResultsExpression of HO-1 was lower in muscle satellite cells isolated from muscles of diabetic db/db mice when compared to their wild-type counterparts, what was accompanied by increased levels of Myf5 or CXCR4, and decreased Mef2 or Pax7. Such cells also displayed diminished differentiation potential when cultured in vitro, as shown by less effective formation of myotubes and reduced expression of myogenic markers (myogenic differentiation antigen - myoD, myogenin and myosin). Blood flow recovery after induction of severe hind limb ischemia was delayed in db/db mice compared to that in normoglycemic individuals. To improve muscle regeneration after ischemia, conditioned media collected from differentiating C2C12 cells (control and HO-1 overexpressing) were injected into hind limbs of diabetic mice. Analysis of blood flow revealed that media from HO-1 overexpressing cells accelerated blood-flow recovery, while immunohistochemical staining assessment of vessel density in injected muscle confirmed increased angiogenesis. The effect might be mediated by stromal-cell derived factor-1α proangiogenic factor, as its secretion is elevated in HO-1 overexpressing cells.ConclusionsIn conclusion, paracrine stimulation of angiogenesis in ischemic skeletal muscle using conditioned media may be a safe approach exploiting protective and proangiogenic properties of HO-1 in diabetes.
Highlights
Diabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties
Expression of HO-1 was lower in muscle satellite cells isolated from muscles of diabetic db/db mice when compared to their wild-type counterparts, what was accompanied by increased levels of Myf5 or CXCR4, and decreased Mef2 or Pax7
Disrupted expression of myogenic markers in muscle satellite cells isolated from diabetic mice Primary mSCs isolated from wild-type and db/db mice showed the same morphology in a routine in vitro culture, but different growth characteristics; db/db cells reached confluence a few days earlier than their wild-type counterparts, they formed elongated tubes less often in a routine cell culture (Figure 1A)
Summary
Diabetes is associated with reduced expression of heme oxygenase-1 (HO-1), a heme-degrading enzyme with cytoprotective and proangiogenic properties. In myoblasts and muscle satellite cells HO-1 improves survival, proliferation and production of proangiogenic growth factors. We aimed to examine whether conditioned media from the HO-1 overexpressing myoblast cell line can improve a blood-flow recovery in ischemic muscles of diabetic mice. After injection of murine myoblasts (C2C12 cell line) overexpressing HO-1 into gastrocnemius muscle of immunocompromized mice, the viability and proliferation of the transplanted cells were improved but their differentiation and formation of myofibers were blocked. HO-1 acts as a cytoprotective and proangiogenic protein which could be targeted to facilitate vascularization of ischemic muscle, especially in diabetic patients characterized by oxidative stress and impaired angiogenesis, there is a need to elaborate a therapeutic protocol that can minimize the risk of adverse effects
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