Role of Runx2 in Calcific Aortic Valve Disease in Mouse Models.

Background: Calcific aortic valve disease is common in the aging population and is characterized by the histological changes of the aortic valves including extracellular matrix remodeling, osteochondrogenic differentiation, and calcification. Combined, these changes lead to aortic sclerosis, aortic stenosis (AS), and eventually to heart failure. Runt-related transcription factor 2 (Runx2) is a transcription factor highly expressed in the calcified aortic valves. However, its definitive role in the progression of calcific aortic valve disease (CAVD) has not been determined. In this study, we utilized constitutive and transient conditional knockout mouse models to assess the molecular, histological, and functional changes in the aortic valve due to Runx2 depletion. Methods: Lineage tracing studies were performed to determine the provenance of the cells giving rise to Runx2+ osteochondrogenic cells in the aortic valves of LDLr-/- mice. Hyperlipidemic mice with a constitutive or temporal depletion of Runx2 in the activated valvular interstitial cells (aVICs) and sinus wall cells were further investigated. Following feeding with a diabetogenic diet, the mice were examined for changes in gene expression, blood flow dynamics, calcification, and histology. Results: The aVICs and sinus wall cells gave rise to Runx2+ osteochondrogenic cells in diseased mouse aortic valves. The conditional depletion of Runx2 in the SM22α+ aVICs and sinus wall cells led to the decreased osteochondrogenic gene expression in diabetic LDLr-/- mice. The transient conditional depletion of Runx2 in the aVICs and sinus wall cells of LDLr-/-ApoB100 CAVD mice early in disease led to a significant reduction in the aortic peak velocity, mean velocity, and mean gradient, suggesting the causal role of Runx2 on the progression of AS. Finally, the leaflet hinge and sinus wall calcification were significantly decreased in the aortic valve following the conditional and temporal Runx2 depletion, but no significant effect on the valve cusp calcification or thickness was observed. Conclusions: In the aortic valve disease, Runx2 was expressed early and was required for the osteochondrogenic differentiation of the aVICs and sinus wall cells. The transient depletion of Runx2 in the aVICs and sinus wall cells in a mouse model of CAVD with a high prevalence of hemodynamic valve dysfunction led to an improved aortic valve function. Our studies also suggest that leaflet hinge and sinus wall calcification, even in the absence of significant leaflet cusp calcification, may be sufficient to cause significant valve dysfunctions in mice.

Conditional Lpar1 gene targeting identifies cell types mediating neuropathic pain.

LPA1 is one of six known receptors (LPA1-6) for lysophosphatidic acid (LPA). Constitutive Lpar1 null mutant mice have been instrumental in identifying roles for LPA-LPA1 signaling in neurobiological processes, brain development, and behavior, as well as modeling human neurological diseases like neuropathic pain. Constitutive Lpar1 null mutant mice are protected from partial sciatic nerve ligation (PSNL)-induced neuropathic pain, however, the cell types that are functionally responsible for mediating this protective effect are unknown. Here, we report the generation of an Lpar1flox/flox conditional null mutant mouse that allows for cre-mediated conditional deletion, combined with a PSNL pain model. Lpar1flox/flox mice were crossed with cre transgenic lines driven by neural gene promoters for nestin (all neural cells), synapsin (neurons), or P0 (Schwann cells). CD11b-cre transgenic mice were also used to delete Lpar1 in microglia. PSNL-initiated pain responses were reduced following cre-mediated Lpar1 deletion with all three neural promoters as well as the CD11b promoter, supporting involvement of Schwann cells, central and/or peripheral neurons, and microglia in mediating pain. Interestingly, rescue responses were nonidentical, implicating distinct roles for Lpar1-expressing cell types. Our results with a new Lpar1 conditional mouse mutant expand an understanding of LPA1 signaling in the PSNL model of neuropathic pain.

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation.

AIMS: Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro. A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE-/- mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size. METHODS AND RESULTS: We used an improved targeting construct to generate LDLr-/- mice with floxed-Runx2 alleles ( LDLr-/- :Runx2 f/f ) such that Cre-mediated recombination ( LDLr-/- :Runx2 ΔSM ) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both LDLr-/- :Runx2 f/f and LDLr-/- :Runx2 ΔSM mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of LDLr-/- :Runx2 ΔSM mice compared to LDLr-/- :Runx2 f/f counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced. CONCLUSIONS: SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression.

Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice.

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2(f/f)) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22-recombinase transgenic allele mice (Runx2(ΔSM)) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2(f/f) mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D-treated Runx2(ΔSM) mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2(f/f) SMCs calcified to a much greater extent than those derived from Runx2(ΔSM) mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients.

Lysophosphatidic acid (LPA) and its receptor, LPA1 , influence embryonic schwann cell migration, myelination, and cell-to-axon segregation.

Schwann cell (SC) migration is an important step preceding myelination and remyelination in the peripheral nervous system, and can be promoted by peptide factors like neuregulins. Here we present evidence that a lipid factor, lysophosphatidic acid (LPA), influences both SC migration and peripheral myelination through its cognate G protein-coupled receptor (GPCR) known as LPA1 . Ultrastructural analyses of peripheral nerves in mouse null-mutants for LPA1 showed delayed SC-to-axon segregation, polyaxonal myelination by single SCs, and thinner myelin sheaths. In primary cultures, LPA promoted SC migration through LPA1 , while analysis of conditioned media from purified dorsal root ganglia neurons using HPLC/MS supported the production of LPA by these neurons. The heterotrimeric G-alpha protein, Gαi , and the small GTPase, Rac1, were identified as important downstream signaling components of LPA1 . These results identify receptor mediated LPA signaling between neurons and SCs that promote SC migration and contribute to the normal development of peripheral nerves through effects on SC-axon segregation and myelination.

Targeted deletion of LPA5 identifies novel roles for lysophosphatidic acid signaling in development of neuropathic pain.

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).

Lysophosphatidic acid signaling may initiate fetal hydrocephalus.

Fetal hydrocephalus (FH), characterized by the accumulation of cerebrospinal fluid, an enlarged head, and neurological dysfunction, is one of the most common neurological disorders of newborns. Although the etiology of FH remains unclear, it is associated with intracranial hemorrhage. Here, we report that lysophosphatidic acid (LPA), a blood-borne lipid that activates signaling through heterotrimeric guanosine 5'-triphosphate-binding protein (G protein)-coupled receptors, provides a molecular explanation for FH associated with hemorrhage. A mouse model of intracranial hemorrhage in which the brains of mouse embryos were exposed to blood or LPA resulted in development of FH. FH development was dependent on the expression of the LPA(1) receptor by neural progenitor cells. Administration of an LPA(1) receptor antagonist blocked development of FH. These findings implicate the LPA signaling pathway in the etiology of FH and suggest new potential targets for developing new treatments for FH.

Lysophosphatidic acid (LPA) receptors: signaling properties and disease relevance.

Lysophosphatidic acid (LPA), a water-soluble phospholipid, has gained significant attention in recent years since the discovery that it acts as a potent signaling molecule with wide-ranging effects on many different target tissues. There are currently five identified G protein-coupled receptors for LPA and more are undergoing validation. The complexity of the expression pattern and signaling properties of LPA receptors results in multiple influences on developmental, physiological, and pathological processes. This review provides a summary of LPA receptor signaling and current views on the potential involvement of this pathway in human diseases that include cardiovascular, cancer, neuropathic pain, neuropsychiatric disorders, reproductive disorders, and fibrosis. The involvement of LPA signaling in these processes implicates multiple, potential drug targets including LPA receptor subtypes and LPA metabolizing enzymes. Modulation of LPA signaling may thus provide therapeutic inroads for the treatment of human disease.

LPA receptors: subtypes and biological actions.

Lysophosphatidic acid (LPA) is a small, ubiquitous phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five known G protein-coupled receptors (GPCRs): LPA(1)-LPA(5). They are encoded by distinct genes named LPAR1-LPAR5 in humans and Lpar1-Lpar5 in mice. The biological roles of LPA are diverse and include developmental, physiological, and pathophysiological effects. This diversity is mediated by broad and overlapping expression patterns and multiple downstream signaling pathways activated by cognate LPA receptors. Studies using cloned receptors and genetic knockout mice have been instrumental in uncovering the significance of this signaling system, notably involving basic cellular processes as well as multiple organ systems such as the nervous system. This has further provided valuable proof-of-concept data to support LPA receptors and LPA metabolic enzymes as targets for the treatment of medically important diseases that include neuropsychiatric disorders, neuropathic pain, infertility, cardiovascular disease, inflammation, fibrosis, and cancer.

Lysophosphatidic acid-induced interleukin-1 beta expression is mediated through Gi/Rho and the generation of reactive oxygen species in macrophages.

Lysophosphatidic acid (LPA), a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoproteins, is known to regulate inflammation and atherosclerosis by binding to its cognate receptors. In this study, we reported that LPA upregulated interleukin-1 beta (IL-1 beta) expression in mouse J774A.1 macrophages. By using pharmacological inhibitors, it was suggested that G(i)/Rho activation and subsequent reactive oxygen species (ROS) production were involved in IL-1 beta induction. In addition, IL-1 beta induction by LPA was also observed in human primary macrophages. In summary, LPA is involved in the processes of inflammation by affecting macrophage behavior.

Parallel SUMOylation-dependent pathways mediate gene- and signal-specific transrepression by LXRs and PPARgamma.

Transrepression is widely utilized to negatively regulate gene expression, but the mechanisms by which different nuclear receptors effect gene- and signal-specific transrepression programs remain poorly understood. Here, we report the identification of alternative SUMOylation-dependent mechanisms that enable PPARgamma and LXRs to negatively regulate overlapping but distinct subsets of proinflammatory genes. Ligand-dependent conjugation of SUMO2/3 to LXRs or SUMO1 to PPARgamma targets them to promoters of TLR target genes, where they prevent the signal-dependent removal of NCoR corepressor complexes required for transcriptional activation. SUMO1-PPARgamma and SUMO2/3-LXRs inhibit distinct NCoR clearance mechanisms, allowing promoter- and TLR-specific patterns of repression. Mutational analysis and studies of naturally occurring oxysterol ligands indicate that the transactivation and SUMOylation-dependent transrepression activities of LXRs can be independently regulated. These studies define parallel but functionally distinct pathways that are utilized by PPARgamma and LXRs to differentially regulate complex programs of gene expression that control immunity and homeostasis.