br Methods br Results br Discussion Previous work by

Methods
Results
Discussion Previous work by the Hirano group investigated the effect of GIP in murine atherosclerotic models and found infusion of GIP (1–42) by osmotic mini-pumps to decrease lesion size after 4 weeks in non-diabetic as well as in diabetic ApoE−/− mice [8], [9]. Mechanistically, the authors found GIP treatment to reduce plaque macrophage content and direct inhibitory effects on foam cell formation mediated via the GIP-receptor in diabetic ApoE−/− mice. However, it remains unclear whether GIP affects plaque morphology and stability and what is the underlying mechanism. Consistent with the previous study in diabetic ApoE−/− mice [9], we found a reduction in lesion macrophages after GIP overexpression in non-diabetic ApoE−/− mice. This effect could be mediated, in general, by a reduction in blood monocytes due to diminished myelopoiesis, changes in systemic or plaque cyto- and chemokine levels or altered chemokine receptor surface expression. Since recent work by Mantelmacher et al. found GIP to increase bone marrow hematopoiesis with the consequence of elevated numbers of differentiated blood leukocytes in mice [15], we speculate that changes in blood monocytes might not explain our phenotype. Here we found GIP to decrease hepatic proinflammatory cyto- and chemokine levels and to directly inhibit CCR2 discover this in monocytes, which was linked to blocked MCP-1-induced monocyte migration In vitro. Based on these results, we speculate that both direct inhibition of CCR2 expression on monocytes as well as systemic (hepatic) reduction of cyto- and chemokine expression might contribute to GIP-mediated reduction of plaque macrophages. However, compared to previous work [8], [9], we could not recapitulate a reduction of atherosclerotic lesion size by chronic GIP treatment, which might be attributable to different methodical approaches and treatment duration (osmotic mini-pumps and 4 weeks treatment vs. viral overexpression and 12 week treatment in our study). However, we demonstrated that GIP improves characteristics of plaque stability (reduced macrophage content and increased collagen content per lesion vs. control), which was linked to inhibitory effects of GIP on monocyte migration in vitro. The significant association between lesion macrophage and collagen content suggests that GIP might increase plaque stability by direct inhibition of plaque inflammation. Consistently, we found less MMP-9 production and a reduction of MMP-9 enzymatic activity after GIP treatment in macrophages In vitro. Mechanistically GIP non-selectively prevented endotoxin-induced activation of NF-κB, JNK, ERK, and p38 signaling pathways in macrophages. Future experimental studies are needed to better understand the exact molecular mechanism of GIP dependent modulation of proinflammatory pathways. In the clinical part of the study we found a positive association between GIP serum levels and PAD as a manifestation of systemic atherosclerosis. In contrast, we did not find association of GIP with CAD. This might be explained by PAD being a strong indicator for advanced atherosclerotic disease, which is linked to worse prognosis relative to CAD patients [16]. These findings seem surprising given the vasoprotective effects of GIP in experimental atherosclerosis. Furthermore under physiological conditions GIP is secreted by intestinal K-cells [1] mainly following food intake. Since recent work demonstrated that GIP secretion can also be induced by inflammatory stimuli like IL-1, IL-6, and LPS independent from food intake [17], [18], we questioned whether the increase in GIP in our patient cohort was linked to inflammatory markers. However, we were unable to detect a significant association between GIP levels and the inflammatory markers CRP and WBC (Supplemental Table 1), making this an unlikely explanation. Interestingly, in a previous study by Nogi et al. [9], the authors found GIP-receptor mRNA expression to be reduced in diabetic vs. non-diabetic mice. Therefore, one might speculate that elevated GIP levels in patients with PAD are a result of reduced GIP-receptor expression in these patients, which will require further investigations. Additional studies are needed to clarify the underlying mechanism of elevated GIP levels in human systemic atherosclerosis.