Atherosclerosis is a complicated cardiovascular disease that stems from prolonged chronic inflammation. This inflammation causes and is also the result of activated monocyte/macrophage infiltration through arterial walls which eventually leads to a necrotic core plaque that narrows the lumen and may lead to catastrophic rupture and death. Vitamin D is known to invoke an anti-inflammatory program in cultured macrophages by decreasing factors such as IL-1β and TNFα. Vitamin D activation to calcitriol (1,25(OH)2D3), occurs mainly in the kidney by the CYP27B1 enzyme, however, nonrenal target cells (NRTCs), such as the macrophage, express low levels of this enzyme as well, and therefore may produce calcitriol at low levels, locally. This NRTC produced calcitriol is hypothesized to reduce inflammation and positively impact disease progression. Importantly though, these mechanisms remain very poorly understood. In humans, the interpretation of local production of calcitriol has been confounded by the abundant levels of endocrine calcitriol produced by the kidney. We aim to eliminate these confounding issues by defining the production, features, biological impact and anti-disease potential of local activity in the absence of endocrine calcitriol by utilizing a unique mouse model we have created wherein the endocrine production of calcitriol in the kidney and thus blood levels of the endocrine hormone are highly deficient. Our use of this mouse model will enable us to explore key details of the importance of 25(OH)D3 uptake into NRTC tissues, the levels of calcitriol that are produced in these tissues, and the biological impact of this production in genetically modified mice that are sensitive to high fat diet-induced chronic inflammation and atherosclerosis. Based on the unique opportunity to separate the endocrine production of calcitriol from the NRTC, the overall hypothesis for this research study is that production of locally-acting calcitriol through cell-specific Cyp27b1 genomic enhancers can ameliorate the chronic inflammation associated with a high fat diet and atherosclerotic plaque formation. We will test this hypothesis by first establishing core principles of NRTC calcitriol production through AIM 1: exploiting our endocrine-deficient Cyp27b1 pseudo-null mouse model to investigate the impact of both 25(OH)D3 (substrate) status and vitamin D nutritional supplementation on NRTC synthesis of calcitriol and its genetic local impact, AIM 2: defining the NRTC- regulatory module (NRTC-RM) for Cyp27b1 expression that is functionally distinct from that of the kidney endocrine module and determine the mechanisms, epigenetics, and tissue-specific factors involved, and finally AIM 3: examining the nonrenal production of calcitriol and its impact on inflammation and on atherosclerotic plaque formation and calcification in Ldlr-/- mice in the disease state of high fat diet-induced chronic inflammation. This knowledge will provide support for nonrenal calci...