Macrophage (MΦ) dysregulation is increasingly becoming recognized as a risk factor for a number of inflammatory complications including atherosclerosis, cancer, and the host response elicited by biomedical devices. It is still unclear what roles the pro-inflammatory (M1) MΦ and pro-healing (M2) MΦ phenotypes play during the healing process. However, it has been shown that a local overabundance of M1 MΦs can potentially lead to a chronically inflamed state of the tissue; while a local over-exuberant M2 MΦ response can lead to tissue fibrosis and even promote tumorigenesis. These notions strengthen the argument that the tight temporal regulation of this phenotype balance is necessary to promote inflammatory resolution that leads to tissue homeostasis. In this study, we have engineered pro-inflammatory MΦ, MΦ-cTLR4 cells, which can be activated to a M1-like MΦ phenotype with a small molecule, the chemical inducer of dimerization (CID) drug. The MΦ-cTLR4 cells when activated with the CID drug, express increased levels of TNFα, IL-6, and iNOS. Activated MΦ-cTLR4 cells stay stimulated for at least 48 hours; once the CID drug is withdrawn, the MΦ-cTLR4 cells return to baseline state within 18 hours. Further, in vitro CID-activated MΦ-cTLR4 cells induce upregulation of VCAM-1 and ICAM-1 on endothelial cells (EC) in a TNFα-dependent manner. With the ability to specifically modulate the MΦ-cTLR4 cells with the presence or absence of a small molecule, we now have the tool necessary to observe a primarily M1 MΦ response during inflammation. By isolating this phase of the wound healing response, it may be possible to determine conditions for ideal healing.

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Vascular calcification, the abnormal deposition of calcium phosphate salts in the vasculature, is associated with a heightened risk of cardiovascular disease in patients with chronic kidney disease and diabetes mellitus. Through a genetic fate mapping strategy, our laboratory has identified vascular smooth muscle cells (SMCs) as a major cell source contributing to bone- and cartilage-like cells in calcifying arteries. Runx2, a transcription factor critical for osteoblast and hypertrophic chondrocyte differentiation, is upregulated in the early stages of a wide range of calcific arterial diseases. Treating SMCs in vitro with elevated phosphate resulted in an increase in phosphorylated Runx2 at serine 319, while total Runx2 remained unaffected. We also found that ERK1/2 signaling and Runx2 were required for SMC osteogenic differentiation and calcification in vitro . To further explore whether Runx2 phosphorylation and activation are critical to Runx2 function in vascular calcification, we mutated Runx2 at serine 301 and 319, the sites phosphorylated by active ERK and shown to be crucial for Runx2 transcriptional activity in osteoblasts, to create ERK-resistant (S310A/S319A) and constitutively active (S301E/S319E) variants. We have generated Runx2 knockout SMCs from Runx2- flox/flox mice carrying SM22α-Cre recombination transgenic allele. Wild type Runx2 and its mutants were introduced into these cells retrovirally. Preliminary Runx2 functional analyses suggest a successful introduction of the constructs and a phosphate-dependent activation of Runx2. Experiments to address a role of Runx2 phosphorylation by ERK in SMC osteogenic differentiation and matrix calcification are ongoing. In conclusion, our current findings along with the ongoing experiments will provide important inside into mechanisms of SMC osteochondrogenic differentiation and vascular calcification, findings that may serve as basis for the development of appropriate therapeutic strategies for this devastating complication.