A purinergic P2Y6 receptor agonist prodrug modulates airway inflammation, remodeling, and hyperreactivity in a mouse model of asthma.

BACKGROUND: Purinergic receptors control cell proliferation, apoptosis, migration, inflammation, and cytokine secretion. Increased expression of specific purinergic receptors is reported in asthma. The role of purinergic P2Y6 receptors (P2Y6R) in asthma is controversial. HYPOTHESIS: P2Y6R activation in asthma improves pulmonary function and reduces inflammation and smooth muscle amount. METHODS: Female mice (C57/BL6, age 30 days) were randomly assigned to receive intranasal house dust mite (HDM) antigen (40 or 80 µg) or saline, 5 days/week, for 6 weeks. Randomly selected subgroups received intraperitoneal P2Y6R agonist prodrug (GC021109; 10 or 100 µg/kg weight/dose) simultaneously with HDM. After 6 weeks, lung function was measured. Lung lavage fluid (LLF) was used to measure total cell count, total protein, and cytokines. Immunohistochemistry for alpha smooth muscle actin (α-SMA) was done. Airway wall thickness was measured on micro-computed tomography (micro-CT) images. RESULTS: Pulmonary function testing revealed a HDM dose-dependent airway hyperresponsiveness. Airway resistance was increased 2-fold while compliance was decreased by 50% at the higher HDM dose (P<0.05). GC021109 prevented these changes. HDM-exposed mice had elevated inflammatory cell and total protein levels in LLF which were prevented by GC021109 (P<0.05). HDM mice also had elevated LLF levels of interleukin (IL)-4, IL-5, IL-12, granulocyte colony stimulating factor, chemokine (C-X-C) motif ligand 1, and leukemia inhibitory factor that were reduced by GC021109 with a dose-dependent pattern. HDM mice had increased peribronchial and perivascular inflammatory cell infiltration and increased α-SMA; these changes were absent with GC021109. Airway wall thickness measured on micro-CT images was increased after HDM exposure and significantly reduced by GC021109 treatment. CONCLUSION: The P2Y6R prodrug GC021109 inhibited allergen-induced changes in pulmonary function, inflammatory responses, and airway and vascular smooth muscle mass. P2Y6R activation may be an effective therapeutic maintenance strategy in asthma.

CCN5 in alveolar epithelial proliferation and differentiation during neonatal lung oxygen injury.

Lung immaturity is the major cause of morbidity and mortality in premature infants, especially those born <28 weeks of gestation. These infants are at high risk of developing respiratory distress syndrome (RDS), a lung disease caused by insufficient surfactant production and immaturity of saccular/alveolar type II epithelial cells in the lung. RDS treatment includes oxygen and respiratory support that improve survival but also increase the risk for bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolarization, airway hyperreactivity, and pulmonary hypertension. The mechanisms regulating normal alveolar development and how injury disrupts normal development to cause BPD are not well understood. We examined the role of the matricellular protein CCN5 (Cysteine-rich protein 61/Connective tissue growth factor/Nephroblastoma-overexpressed protein) in the development of BPD. Cultured non-proliferating alveolar type II cells expressed low levels of CCN5 protein, and displayed higher levels during proliferation. siRNA targeting of CCN5 reduced alveolar type II cell proliferation and migration in cell culture. In a mouse model of hyperoxia-induced BPD, CCN5 protein was increased only in proliferating alveolar type I cells. Alveolar epithelial cells co-expressing markers of type II cells and type I cells also appeared. The results suggest that hyperoxic injury in immature lungs induces proliferation of type I cells and trans-differentiation of type II cells into type I cells. We propose that the mechanism of the injury response in BPD includes CCN5 expression. Study of CCN5 in neonatal alveolar injury will further our understanding of BPD pathophysiology while providing a mechanistic foundation for therapeutic approaches.

Matrix production and remodeling as therapeutic targets for uterine leiomyoma.

Uterine leiomyoma, commonly known as fibroids, is a benign neoplasm of smooth muscle in women. The incidence of clinically symptomatic fibroids in reproductive-age women is approximately 20 %, with nearly 80 % of black women suffering from this condition. Symptoms include severe pain and hemorrhage; fibroids are also a major cause of infertility or sub-fertility in women. Uterine leiomyoma consist of hyperplastic smooth muscle cells and an excess deposition of extracellular matrix, specifically collagen, fibronectin, and sulfated proteoglycans. Extracellular matrix components interact and signal through integrin-ß1 on the surface of uterine leiomyoma smooth muscle cells, provide growth factor storage, and act as co-receptors for growth factor-receptor binding. ECM and growth factor signaling through integrin-ß1 and growth factor receptors significantly increases cell proliferation and ECM deposition in uterine leiomyoma. Growth factors TGF-ß, IGF, PDGF, FGF and EGF are all shown to promote uterine leiomyoma progression and signal through multiple pathways to increase the expression of genes encoding matrix or matrix-modifying proteins. Decreasing integrin expression, reducing growth factor action and inhibiting ECM action on uterine leiomyoma smooth muscle cells are important opportunities to treat uterine leiomyoma without use of the current surgical procedures. Both natural compounds and chemicals are shown to decrease fibrosis and uterine leiomyoma progression, but further analysis is needed to make inroads in treating this common women's health issue.

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation.

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.

CCN5: biology and pathophysiology.

CCN5 is one of six proteins in the CCN family. This family of proteins has been shown to play important roles in many processes, including proliferation, migration, adhesion, extracellular matrix regulation, angiogenesis, tumorigenesis, fibrosis, and implantation. In this review, we focus on the biological and putative pathophysiological roles of CCN5. This intriguing protein is structurally unique among the CCN family members, and has a unique biological activity profile as well.

Peroxisome proliferator-activated receptor alpha is crucial for iloprost-induced in vivo angiogenesis and vascular endothelial growth factor upregulation.

We have previously demonstrated that iloprost, a stable prostacyclin (PGI(2)) analogue, induces angiogenesis in vivo, through a vascular endothelial growth factor (VEGF)-dependent mechanism. In this study, we demonstrate that iloprost-induced angiogenesis and VEGF upregulation are modulated by peroxisome proliferator-activated receptor-alpha (PPARalpha), a ligand-inducible transcription factor that belongs to the nuclear hormone receptor superfamily and plays multiple biological activities in the vascular system. We show that iloprost is unable to induce angiogenesis in mice lacking the PPARalpha gene (PPARalpha-/- mice). Likewise, iloprost-induced VEGF upregulation is absent in PPARalpha-/- mice. In contrast, iloprost induces a robust angiogenic response in wild-type mice, along with local upregulation of VEGF. Importantly, mice lacking the PPARalpha gene exhibit a normal angiogenic response to VEGF, indicating that the absence of PPARalpha does not result in a general impairment of angiogenesis, but specifically affects the ability of iloprost to induce angiogenesis. Our data demonstrate unexpected functional relationships between the PGI(2) system and the PPAR signaling pathway and shed new light on the molecular mechanisms involved in iloprost-induced angiogenesis.

Selective activation of peroxisome proliferator-activated receptor (PPAR)alpha and PPAR gamma induces neoangiogenesis through a vascular endothelial growth factor-dependent mechanism.

OBJECTIVE: Peroxisome proliferator-activated receptors (PPARs) are therapeutic targets for fibrates and thiazolidinediones, which are commonly used to ameliorate hyperlipidemia and hyperglycemia in type 2 diabetes. In this study, we evaluated whether activation of PPAR alpha and PPAR gamma stimulates neoangiogenesis. RESEARCH DESIGN AND METHODS: We used selective synthetic PPAR alpha and PPAR gamma agonists and investigated their angiogenic potentials in vitro and in vivo. RESULTS: Activation of PPAR alpha and PPAR gamma leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine vascular endothelial growth factor (VEGF). Neovascularization also occurs in vivo, when PPAR alpha and PPAR gamma agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPAR alpha and PPAR gamma agonists are respectively used in PPAR alpha knockout mice and mice treated with a specific PPAR gamma inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPAR alpha- and PPAR gamma-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPAR alpha- and PPAR gamma-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPAR alpha and PPAR gamma agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt. CONCLUSIONS: These findings demonstrate that PPAR alpha and PPAR gamma activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPAR alpha and PPAR gamma agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes.

Impaired clearance of apoptotic cells promotes synergy between atherogenesis and autoimmune disease.

To clarify the link between autoimmune disease and hypercholesterolemia, we created the gld.apoE(-/-) mouse as a model of accelerated atherosclerosis. Atherosclerotic lesion area was significantly increased in gld.apoE(-/-) mice compared with apoE(-/-) mice. gld.apoE(-/-) mice also displayed increases in lymphadenopathy, splenomegaly, and autoantibodies compared with gld mice, and these effects were exacerbated by high cholesterol diet. gld.apoE(-/-) mice exhibited higher levels of apoptotic cells, yet a reduced frequency of engulfed apoptotic nuclei within macrophages. Infusion of lysophosphatidylcholine, a component of oxidized low density lipoprotein, markedly decreased apoptotic cell clearance in gld mice, indicating that hypercholesterolemia promotes autoimmune disease in this background. These data suggest that defects in apoptotic cell clearance promote synergy between atherosclerotic and autoimmune diseases.

The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells.

Uterine fibroids (leiomyomas) are a major women's health problem. Currently, the standard for treatment remains hysterectomy, since no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are benign neoplasias of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by CCN5, a growth arrest-specific gene in vascular smooth muscle cells which is induced and maintained by heparin treatment. Using autologous human myometrial and leiomyoma smooth muscle cells, we demonstrate that the proliferation and motility of both cell types are inhibited by the overexpression of CCN5. Surprisingly, we show that even though CCN5 is induced by heparin in vascular smooth muscle cells, treatment with heparin does not induce CCN5 expression in human uterine smooth muscle cells. Furthermore, we examine CCN5 mRNA expression in 10 autologous pairs of human myometrial and leiomyoma tissues and determine that CCN5 is down-regulated in 100% of the leiomyoma tissues analysed when compared to their normal myometrial counterparts. Thus, our data strongly suggest that CCN5 may exert an important function in maintaining the normal uterine phenotype and that loss of the anti-proliferative protein CCN5 from normal myometrium may account, at least in part, for tumorigenesis.

Estrogen induces CCN5 expression in the rat uterus in vivo.

Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.

CCN5 modulates the antiproliferative effect of heparin and regulates cell motility in vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cell (VSMC) hyperplasia plays an important role in both chronic and acute vascular pathologies including atherosclerosis and restenosis. Considerable work has focused on the mechanisms regulating VSMC proliferation and motility. Earlier work in our lab revealed a novel growth arrest-specific (gas) gene induced in VSMC exposed to the antiproliferative agent heparin. This gene is a member of the CCN family and has been given the name CCN5. The objective of the present study is to elucidate the function of CCN5 protein and to explore its mechanism of action in VSMC. RESULTS: Using RNA interference (RNAi), we first demonstrate that CCN5 is required for the antiproliferative effect of heparin in VSMC. We also use this gene knockdown approach to show that CCN5 is an important negative regulator of motility. To explore the mechanism of action of CCN5 on VSMC motility, we use RNAi to demonstrate that knock down of CCN5 up regulates expression of matrix metalloproteinase-2 (MMP-2), an important stimulator of motility in VSMC. In addition, forced expression of CCN5 via adenovirus results in reduced MMP-2 activity, this also corroborates the gene knock down results. Finally, we show that loss of CCN5 expression in VSMC causes changes in VSMC morphology and cytoskeletal organization, including a reduction in the amount and macromolecular assembly of smooth muscle cell alpha-actin. CONCLUSIONS: This work provides important new insights into the regulation of smooth muscle cell proliferation and motility by CCN5 and may aid the development of therapies for vascular diseases.

Heparin inhibits lipopolysaccharide (LPS) binding to leukocytes and LPS-induced cytokine production.

The glycosaminoglycan heparin is known to exhibit anti-inflammatory properties unrelated to its anticoagulant activity. However, in a generalized inflammatory response with implanted or extracorporeal devices, the beneficial effect of heparin coating and/or systemic administration is still unclear as well as the precise mechanisms of action. In the present study, we have first studied the effect of heparin on lipopolysaccharide (LPS)-induced cytokine production by human blood monocytes. Our results indicated that the production of interleukin-1alpha, tumor necrosis factor-alpha, and interleukin-8 was significantly decreased when heparin was simultaneously incubated with Escherichia coli LPS. Because the modulation of heparin on monocyte activation could be mediated by its binding via CD14, the main LPS receptor on monocytes, we then studied the binding of LPS and heparin to leukocytes from human blood and to Chinese hamster ovary cells transfected with the human CD14 gene. The data by flow cytometry showed the binding of biotinylated heparin to leukocytes. Moreover, the experiments performed on leukocytes and on CD14-positive Chinese hamster ovary cells indicated that heparin inhibited LPS binding. From our results, we conclude that: 1. heparin is an effective inhibitor of LPS-induced monocyte activation, and 2. heparin inhibits the binding of LPS to cells via a CD14-independent pathway. This study suggests a potentially important therapeutic application for heparin or heparin analogs to prevent inflammation with biomaterials.

Heparin inhibits the motility and proliferation of human myometrial and leiomyoma smooth muscle cells.

Uterine fibroids (leiomyomas) are a major women's health problem. Currently, the standard for treatment remains hysterectomy, because no other treatment modalities can reduce both symptoms and recurrence. As leiomyomas are a hyperproliferation of smooth muscle cells, we sought to understand the regulation of uterine smooth muscle cell mitogenesis by the glycosaminoglycan heparin, which has been extensively studied as an anti-proliferative molecule in vascular smooth muscle cells. Using matched pairs of human myometrial and leiomyoma smooth muscle cells from the same uterus, we demonstrate that the proliferation and motility of both cell types are inhibited by heparin. We report that the decrease in cell number seen in the presence of heparin is not because of cell death. Interestingly, there is significant patient-to-patient variability in the proliferation response but not in the motility response to heparin. Furthermore, nonanticoagulant and anticoagulant heparin were equally effective at inhibiting leiomyoma and myometrial smooth muscle cell proliferation. These results warrant further investigation into the possibility that heparin might be useful in the treatment of uterine fibroids.

CCN5 is a growth arrest-specific gene that regulates smooth muscle cell proliferation and motility.

Vascular smooth muscle cell (VSMC) hyperplasia plays an important role in both chronic and acute vascular pathologies. Considerable work has focused on the mechanisms regulating VSMC growth and the search for agents that could suppress VSMC hyperproliferation. One of the several inhibitors studied is the glycosaminoglycan heparin, which inhibits VSMC proliferation and migration both in cell culture and in animal models (Mishra-Gorur K, Delmolino LM, Castellot Jr JJ: Biological functions of heparan sulfate and heparan sulfate proteoglycans. Trends Glycosci Glycotechnol 1998, 10:193-210). To aid our understanding of the anti-proliferative mechanism of action of heparin, we used a subtractive hybridization approach to isolate and characterize a novel growth arrest-specific (gas) gene induced in VSMCs exposed to heparin (Delmolino LM, Stearns NA, Castellot Jr JJ: Heparin induces a member of the CCN family which has characteristics of a growth arrest specific gene. Mol Biol Cell 1997, 8:287a and Delmolino LM, Stearns NA, Castellot Jr JJ: COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells. J Cell Physiol 2001, 188:45-55). This gene is a member of the cysteine-rich 61/connective tissue growth factor/nephroblastoma-overexpressed (CCN) family and has been given the name CCN5. In this report, we provide functional evidence that CCN5 can inhibit VSMC proliferation, motility, and invasiveness. In contrast, adhesion and apoptosis are unaffected by CCN5 in this cell type. We also significantly extend previous data from our laboratory that suggests CCN5 is a growth arrest-specific (gas) gene. Furthermore, we map for the first time the cellular localization of CCN5 protein in cultured VSMCs. We also examine uninjured and balloon-injured rat carotid arteries for CCN5 expression. The results from the in vitro and in vivo localization studies show that CCN5 is temporally and spatially expressed in a manner consistent with a role in regulating proliferation, motility, and invasiveness of VSMCs.

Heparin inhibits phosphorylation and autonomous activity of Ca(2+)/calmodulin-dependent protein kinase II in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca(2+) regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the delta-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca(2+)/calmodulin-independent CaM kinase II activity. This effect was less evident in heparin-resistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Finally, the effects of pharmacological inhibitors of phosphatases like okadaic acid, calyculin, and tautomycin suggest that heparin inhibits CaM kinase II phosphorylation by activating protein phosphatases 1 and 2A. These findings support the hypothesis that alterations in calcium-mediated mitogenic signaling pathways may be involved in the antiproliferative mechanism of action of heparin.