Geert Carmeliet

Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow–derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.

Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)2 vitamin D [1,25(OH)2D3] action on calcium homeostasis. We assessed the rate of serum 45Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D9K expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA1b) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)2D3 injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D9K expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D9K and PMCA1b expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D9K, should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels.

During endochondral bone development, the first osteoblasts differentiate in the perichondrium surrounding avascular cartilaginous rudiments; the source of trabecular osteoblasts inside the later bone is, however, unknown. Here, we generated tamoxifen-inducible transgenic mice bred to Rosa26R-LacZ reporter mice to follow the fates of stage-selective subsets of osteoblast lineage cells. Pulse-chase studies showed that osterix-expressing osteoblast precursors, labeled in the perichondrium prior to vascular invasion of the cartilage, give rise to trabecular osteoblasts, osteocytes, and stromal cells inside the developing bone. Throughout the translocation, some precursors were found to intimately associate with invading blood vessels, in pericyte-like fashion. A similar coinvasion occurs during endochondral healing of bone fractures. In contrast, perichondrial mature osteoblasts did not exhibit perivascular localization and remained in the outer cortex of developing bones. These findings reveal the specific involvement of immature osteoblast precursors in the coupled vascular and osteogenic transformation essential to endochondral bone development and repair.

Vitamin D and cancer

1,25-dihydroxy Vitamin D [1,25-(OH)(2)D] exerts its effects via the vitamin D receptor (VDR) that belongs to the steroid/thyroid hormone receptor superfamily leading to gene regulation which results in various biological responses. Within the last two decades, the receptor has been shown to be present not only in classical target tissues such as bone, kidney and intestine but also in many other non-classical tissues. Besides the almost universal presence of VDRs, some cell types (e.g. keratinocytes, monocytes, bone, placenta) are capable of metabolizing 25-hydroxyvitamin D to 1,25(OH)(2)D by the enzyme 1alpha-hydroxylase (CYP27B1). The combined presence of 25(OH)D-1alpha-hydroxylase as well as the specific receptor in several tissues introduced the idea of a paracrine role for 1,25(OH)(2)D. Moreover, it has been demonstrated that 1,25(OH)(2)D can induce differentiation and inhibit proliferation of a wide variety of cell types. The molecular mechanisms behind this antiproliferative action is thoroughly explored but the whole picture is still difficult to understand. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. However the precise hierarchical structure of this wide diversity of actions of 1,25(OH)(2)D on genes influencing cell cycle progression is not firmly established nor do we understand which pathways are essential and which redundant. The antiproliferative action makes 1,25-(OH)(2)D and its analogs a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. This review focuses on the effects of 1,25(OH)(2)D and its analogs on cell proliferation, the results in in vivo experiments in Vitamin D deficient or resistant animals to cancer and the current epidemiological and intervention studies linking Vitamin D status or treatment and human cancer.

VEGF : A modifier of the del22q11 (DiGeorge) syndrome?

Hemizygous deletion of chromosome 22q11 (del22q11) causes thymic, parathyroid, craniofacial and life-threatening cardiovascular birth defects in 1 in 4,000 infants. The del22q11 syndrome is likely caused by haploinsufficiency of TBX1, but its variable expressivity indicates the involvement of additional modifiers. Here, we report that absence of the Vegf164 isoform caused birth defects in mice, reminiscent of those found in del22q11 patients. The close correlation of birth and vascular defects indicated that vascular dysgenesis may pathogenetically contribute to the birth defects. Vegf interacted with Tbx1, as Tbx1 expression was reduced in Vegf164-deficient embryos and knocked-down vegf levels enhanced the pharyngeal arch artery defects induced by tbx1 knockdown in zebrafish. Moreover, initial evidence suggested that a VEGF promoter haplotype was associated with an increased risk for cardiovascular birth defects in del22q11 individuals. These genetic data in mouse, fish and human indicate that VEGF is a modifier of cardiovascular birth defects in the del22q11 syndrome.

Vitamin D: Metabolism, Molecular Mechanism of Action, and Pleiotropic Effects

1,25-Dihydroxvitamin D3 [1,25(OH)2D3] is the hormonally active form of vitamin D. The genomic mechanism of 1,25(OH)2D3 action involves the direct binding of the 1,25(OH)2D3 activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Numerous VDR co-regulatory proteins have been identified, and genome-wide studies have shown that the actions of 1,25(OH)2D3 involve regulation of gene activity at a range of locations many kilobases from the transcription start site. The structure of the liganded VDR/RXR complex was recently characterized using cryoelectron microscopy, X-ray scattering, and hydrogen deuterium exchange. These recent technological advances will result in a more complete understanding of VDR coactivator interactions, thus facilitating cell and gene specific clinical applications. Although the identification of mechanisms mediating VDR-regulated transcription has been one focus of recent research in the field, other topics of fundamental importance include the identification and functional significance of proteins involved in the metabolism of vitamin D. CYP2R1 has been identified as the most important 25-hydroxylase, and a critical role for CYP24A1 in humans was noted in studies showing that inactivating mutations in CYP24A1 are a probable cause of idiopathic infantile hypercalcemia. In addition, studies using knockout and transgenic mice have provided new insight on the physiological role of vitamin D in classical target tissues as well as evidence of extraskeletal effects of 1,25(OH)2D3 including inhibition of cancer progression, effects on the cardiovascular system, and immunomodulatory effects in certain autoimmune diseases. Some of the mechanistic findings in mouse models have also been observed in humans. The identification of similar pathways in humans could lead to the development of new therapies to prevent and treat disease.

The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent

Plasminogen activator inhibitor 1 (PAI‐1) is believed to control proteolytic activity and cell migration during angiogenesis. We previously demonstrated in vivo that this inhibitor is necessary for optimal tumor invasion and vascularization. We also showed that PAI‐1 angiogenic activity is associated with its control of plasminogen activation but not with the regulation of cell‐matrix interaction. To dissect the role of the various components of the plasminogen activation system during angiogenesis, we have adapted the aortic ring assay to use vessels from gene‐inactivated mice. The single deficiency of tPA, uPA, or uPAR, as well as combined deficiencies of uPA and tPA, did not dramatically affect microvessel formation. Deficiency of plasminogen delayed microves‐sel outgrowth. Lack of PAI‐1 completely abolished angio‐genesis, demonstrating its importance in the control of plasmin‐mediated proteolysis. Microvessel outgrowth from PAI‐1‐/‐ aortic rings could be restored by adding exogenous PAI‐1 (wild‐type serum or purified recombi‐nant PAI‐1). Addition of recombinant PAI‐1 led to a bell‐shaped angiogenic response clearly showing that PAI‐1 is proangiogenic at physiological concentrations and antiangiogenic at higher levels. Using specific PAI‐1 mutants, we could demonstrate that PAI‐1 promotes an‐giogenesis at physiological (nanomolar) concentrations through its antiproteolytic activity rather than by interacting with vitronectin.—Devy, L., Blacher, S., Grignet‐Debrus, C., Bajou, K., Masson, V., Gerard, R. D., Gils, A., Carmeliet, G., Carmeliet, P., Declerck, P. J., Noèl, A., Foidart, J. M. The pro‐ or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 16, 147–154 (2002)

Normocalcemia is maintained in mice under conditions of calcium malabsorption by vitamin D-induced inhibition of bone mineralization

Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D [1,25(OH)(2)D], which can elicit calcium mobilization from bone when intestinal calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.

Vitamin D receptor in chondrocytes promotes osteoclastogenesis and regulates FGF23 production in osteoblasts

Genomic actions induced by 1alpha25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are crucial for normal bone metabolism, mainly because they regulate active intestinal calcium transport. To evaluate whether the vitamin D receptor (VDR) has a specific role in growth-plate development and endochondral bone formation, we investigated mice with conditional inactivation of VDR in chondrocytes. Growth-plate chondrocyte development was not affected by the lack of VDR. Yet vascular invasion was impaired, and osteoclast number was reduced in juvenile mice, resulting in increased trabecular bone mass. In vitro experiments confirmed that VDR signaling in chondrocytes directly regulated osteoclastogenesis by inducing receptor activator of NF-kappaB ligand (RANKL) expression. Remarkably, mineral homeostasis was also affected in chondrocyte-specific VDR-null mice, as serum phosphate and 1,25(OH)(2)D levels were increased in young mice, in whom growth-plate activity is important. Both in vivo and in vitro analysis indicated that VDR inactivation in chondrocytes reduced the expression of FGF23 by osteoblasts and consequently led to increased renal expression of 1alpha-hydroxylase and of sodium phosphate cotransporter type IIa. Taken together, our findings provide evidence that VDR signaling in chondrocytes is required for timely osteoclast formation during bone development and for the endocrine action of bone in phosphate homeostasis.