HMGB1-Driven Inflammation and Intimal Hyperplasia After Arterial Injury Involves Cell-Specific Actions Mediated by TLR4

Abstract

Objective Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. Approach and Results Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2–toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. Conclusions These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule–mediated vascular inflammatory responses.

Cathepsin L Promotes Vascular Intimal Hyperplasia after Arterial Injury

“…We have previously shown that TLR4 on myeloid cells plays a key role in the development of IH in the carotid artery wire injury model (12). Here, we show that myeloid-specific deletion of TLR4 or its downstream signaling adapter protein, MyD88, significantly prevented the increases in cathepsin L mRNA levels observed 3 d after wire injury ( Figures 2A, B, C).…”

“…Here, we show that myeloid-specific deletion of TLR4 or its downstream signaling adapter protein, MyD88, significantly prevented the increases in cathepsin L mRNA levels observed 3 d after wire injury ( Figures 2A, B, C). Although we had previously shown that global MyD88 deletion suppresses intimal hyperplasia (12), we now show that myeloid-specific MyD88 deletion also tissue lysates were added to a 96-well plate and 50 μl of CL reaction buffer was added to each sample and 2 μl of the 10 mM Ac-FR-AFC substrate was added to each well. These were incubated at 37°C for 1-2 h. Each sample was read by a bottom-reading fluorometer at excitation/emission wavelengths of 400/505 nm.…”

“…We (12) and others (15)(16)(17) have shown that TLR4/MyD88 signaling is significantly involved in IH after arterial injury. We show here that cathepsin L expression is markedly increased after carotid artery wire injury and that this depends muscle cell (SMC) accumulation and extracellular matrix deposition, resulting in intimal hyperplasia (IH) and vessel or stent occlusion (2)(3)(4).…”

“…Measurements included luminal area, medial area, intimal area, vessel area, and the lengths of the internal elastic lamina and external elastic lamina. The intima-to-media (I/M) ratio was calculated as previously described (12). All sections were analyzed separately by two investigators blinded to the study design.…”

“…Mouse cathepsin L is most homologous to human cathepsin V (10,11). Since macrophage infiltration and activation are critical steps in the IH that follows acute injury to the arterial wall (12)(13)(14), we postulate that cathepsin L contributes to IH and that the TLR4-MyD88 pathway in myeloid lineages regulates cathepsin L expression after wire injury.…”