HMGB1 and TLR4 mediate skeletal muscle recovery in a murine model of hindlimb ischemia

Abstract

Introduction We have previously shown that the danger signal High Mobility Group Box 1 (HMGB1) promotes angiogenesis when administered to ischemic muscle. HMGB1 signals through Toll-like receptor 4 (TLR4) as well as the receptor for advanced glycation end-products (RAGE). However, the actions of these receptors in ischemic injury and muscle recovery are not known. We hypothesize that TLR4 mediates tissue recovery and angiogenesis in response to ischemia. Methods Femoral artery ligation was performed in control, TLR4 competent (C3H/HeOuJ), and incompetent (C3H/HeJ) mice, as well as RAGE knockout mice and their C57B6 control counterparts. In other experiments, control mice were pretreated with anti-HMGBI neutralizing antibody before femoral artery ligation. After two weeks, limb perfusion was evaluated using laser Doppler perfusion imaging (LDPI) and reported as the ratio of blood flow in the ischemic to nonischemic limb. Muscle necrosis, fat replacement, and vascular density in the anterior tibialis muscle were quantified histologically. In vitro, TLR4 and RAGE expression was evaluated in human dermal microvascular endothelial cells (HDMVECs) in response to hypoxia. HDMVECs treated with HMGB1 alone and in the presence of anti-TLR4 antibody were probed for phosphorylated ERK (p-ERK), a signaling molecule critical to EC angiogenic behavior. Results Both anti-HMGB1 antibody as well as defective TLR4 signaling in HeJ mice resulted in prominent muscle necrosis two weeks after femoral artery ligation. Control HeOuJ mice had less necrosis than TLR4 incompetent HeJ mice, but a greater amount of fat replacement. In contrast to control C3H mice, control C57B6 mice demonstrated prominent muscle regeneration with very little necrosis. Muscle regeneration was not dependent on RAGE. While vascular density did not differ between strains, mice with intact RAGE and TLR4 signaling had less blood flow in ischemic limbs compared to mutant strains. In vitro, EC TLR4 expression increased in response to hypoxia while TLR4 antagonism decreased HMGB1-induced activation of ERK. Conclusion Both HMGB1 and TLR4 protect against muscle necrosis after hindlimb ischemia. However, muscle regeneration does not appear to be tied to vascular density. HMGB1 likely activates angiogenic behavior in EC in vitro, and this activation may be modulated by TLR4. The improvement in blood flow seen in mice with absent TLR4 and RAGE signaling may suggest anti-angiogenic roles for both receptors, or vasoconstriction induced by TLR4 and RAGE mediated inflammatory pathways.

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

“…Whereas most have focused on the identification and testing of proangiogenic compounds or cellular therapies, there has been much less emphasis on evaluating the role of modulating inflammation to improve tissue recovery from the ischemic insult. We and others have investigated the role of toll-like receptor (TLR) activation in promoting angiogenesis and muscle recovery in a murine model of hindlimb ischemia (7)(8)(9)(10)(11)(12)(13)(14). TLRs mediate innate immune responses to bacterial components, including gram-negative bacterial lipopolysaccharide (LPS) (TLR4) and gram-positive bacterial lipoproteins (TLR2) (15)(16)(17)(18).…”

“…TLRs also sense endogenously released damageassociated molecular patterns (DAMPS), such as high-mobility group box-1 (HMGB1) to initiate an immune response (19). We have previously demonstrated that HMGB1, TLR4 and TLR2 modulate inflammation and muscle recovery after ischemia (8)(9)(10). TLR2 knockout (TLR2KO) mice develop abnormal vasculature, possibly influencing the regenerative process, whereas TLR4KO mice exhibit a robust regenerative phenotype.…”

CD13 Restricts TLR4 Endocytic Signal Transduction in Inflammation

“…In response to infection, the absence of TLR4 activation results in death because of septicemia, whereas excessive activation can lead to serious complications of dysregulated inflammation and further tissue damage. Similarly, repair of sterile tissue damage also requires a well-controlled innate immune response in diseases such as myocardial infarction, stroke, and peripheral artery disease (1)(2)(3). Therefore, tight control of TLR4 signaling is imperative for a balanced and effective immune response.…”

High-mobility group box-1 and its role in angiogenesis

“…Indeed, as a DAMP molecule, HMGB1 gets more and more attention as a proangiogenic factor that could modulate the function of these angiogenesis-related cells. Upregulation of HMGB1 has been reported in many angiogenesis-related conditions, including cancer [7], cornea neovascularization [14], PDR [15], wound-healing [16], and ischemiainduced angiogenesis [17]. Here, we describe recent progress on how HMGB1 regulates angiogenesis in physiological or pathological status and discuss the therapeutic potential of targeting HMGB1 in angiogenesis-related conditions.…”