Hitoshi Kashiwagi

Roles of prostanoids in the pathogenesis of cardiovascular diseases: Novel insights from knockout mouse studies

Prostanoids consisting of prostaglandins (PGs) and thromboxanes (TXs) are produced from arachidonic acids, representative fatty acids contained in cell membrane, by the sequential actions of phospholipase A(2), cyclooxygenases and respective prostanoid synthases. Prostanoids are released outside of the cells immediately after biosynthesis and exert a wide range of actions in the body. These actions are mediated by their respective G protein-coupled receptors expressed in the target cells, which receptors include the DP, EP, FP, IP and TP receptors for PGD(2), PGE(2), PGF(2)α, PGI(2) and TXA(2), respectively. In addition, there are four subtypes of the EP receptors: EP(1), EP(2), EP(3) and EP(4). Recently, roles of prostanoids in the pathogenesis of cardiovascular diseases have been widely examined using mice lacking each prostanoid receptor individually or enzyme participating in prostanoid biosynthesis. These studies have revealed important and novel roles of prostanoids in the development of cardiovascular diseases, such as acute myocardial infarction, cardiac hypertrophy, atherosclerosis, vascular remodeling, hypertension and cerebral thrombosis. Roles of prostanoids in the generation of inflammatory tachycardia and the regulation of platelet function have also been clarified. In this review, we summarize these roles of prostanoids revealed from knockout mouse studies.

The intrinsic prostaglandin E2–EP4 system of the renal tubular epithelium limits the development of tubulointerstitial fibrosis in mice

Inflammatory responses in the kidney lead to tubulointerstitial fibrosis, a common feature of chronic kidney diseases. Here we examined the role of prostaglandin E(2) (PGE(2)) in the development of tubulointerstitial fibrosis. In the kidneys of wild-type mice, unilateral ureteral obstruction leads to progressive tubulointerstitial fibrosis with macrophage infiltration and myofibroblast proliferation. This was accompanied by an upregulation of COX-2 and PGE(2) receptor subtype EP(4) mRNAs. In the kidneys of EP(4) gene knockout mice, however, obstruction-induced histological alterations were significantly augmented. In contrast, an EP(4)-specific agonist significantly attenuated these alterations in the kidneys of wild-type mice. The mRNAs for macrophage chemokines and profibrotic growth factors were upregulated in the kidneys of wild-type mice after ureteral obstruction. This was significantly augmented in the kidneys of EP(4)-knockout mice and suppressed by the EP(4) agonist but only in the kidneys of wild-type mice. Notably, COX-2 and MCP-1 proteins, as well as EP(4) mRNA, were localized in renal tubular epithelial cells after ureteral obstruction. In cultured renal fibroblasts, another EP(4)-specific agonist significantly inhibited PDGF-induced proliferation and profibrotic connective tissue growth factor production. Hence, an endogenous PGE(2)-EP(4) system in the tubular epithelium limits the development of tubulointerstitial fibrosis by suppressing inflammatory responses.

Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation

The effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2-/- platelets) and EP4 (EP4-/- platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1-259 and AE1-329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2-/- and EP4-/- platelets. In human platelets, AE1-259 and AE1-329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1-329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1-259. AE1-329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3-208, an EP4 antagonist. In addition, AE1-329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

The Novel Prostaglandin I2 Mimetic ONO-1301 Escapes Desensitization in an Antiplatelet Effect Due to Its Inhibitory Action on Thromboxane A2 Synthesis in Mice

ONO-1301 [(E)-[5-[2-[1-phenyl-1-(3-pyridyl)methylidene-aminooxy]ethyl]-7,8-dihydronaphthalene-1-yloxy]acetic acid] is a novel prostaglandin (PG) I2 mimetic with inhibitory activity on the thromboxane (TX) A2 synthase. Interestingly, ONO-1301 retains its inhibitory effect on platelet aggregation after repeated administration, while beraprost, a representative agonist for the PGI2 receptor (IP), loses its inhibitory effect after repeated administration. In the present study, we intended to clarify the mechanism by which ONO-1301 escapes desensitization of an antiplatelet effect. In platelets prepared from wild-type mice, ONO-1301 inhibited collagen-induced aggregation and stimulated cAMP production in an IP-dependent manner. In addition, ONO-1301 inhibited arachidonic acid–induced TXA2 production in platelets lacking IP. Despite the decrease in stimulatory action on cAMP production, the antiplatelet effect of ONO-1301 hardly changed after repeated administration for 10 days in wild-type mice. Noteworthy, beraprost could retain its antiplatelet effect after repeated administration in combination with a low dose of ozagrel, a TXA2 synthase inhibitor. Therefore, we hypothesized that chronic IP stimulation by beraprost induces an increase in TXA2 production, leading to reduction in the antiplatelet effect. As expected, repeated administration of beraprost increased the plasma and urinary levels of a TXA2 metabolite, while ONO-1301 did not increase them significantly. In addition, beraprost could retain the ability to inhibit platelet aggregation after repeated administration in mice lacking the TXA2 receptor (TP). These results indicate that TP-mediated signaling participates in platelet desensitization against IP agonists and that simultaneous inhibition of TXA2 production confers resistance against desensitization on IP agonists.

Diethylstilbestrol administration inhibits theca cell androgen and granulosa cell estrogen production in immature rat ovary

Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

Prostaglandin I2 suppresses the development of diet-induced nonalcoholic steatohepatitis in mice

Nonalcoholic steatohepatitis (NASH) is a hepatic manifestation of metabolic syndrome. Although the prostaglandin (PG)I2 receptor IP is expressed broadly in the liver, the role of PGI2‐IP signaling in the development of NASH remains to be determined. Here, we investigated the role of the PGI2‐IP system in the development of steatohepatitis using mice lacking the PGI2 receptor IP [IP‐knockout (IP‐KO) mice] and beraprost (BPS), a specific IP agonist. IP‐KO and wild‐type (WT) mice were fed a methionine‐ and choline‐deficient diet (MCDD) for 2, 5, or 10 wk. BPS was administered orally to mice every day during the experimental periods. The effect of BPS on the expression of chemokine and inflammatory cytokines was examined also in cultured Kupffer cells. WT mice fed MCDD developed steatohepatitis at 10 wk. IP‐KO mice developed steatohepatitis at 5 wk with augmented histologic derangements accompanied by increased hepatic monocyte chemoattractant protein‐1 (MCP‐1) and TNF‐α concentrations. After 10 wk of MCDD, IP‐KO mice had greater hepatic iron deposition with prominent oxidative stress, resulting in hepatocyte damage. In WT mice, BPS improved histologic and biochemical parameters of steatohepatitis, accompanied by reduced hepatic concentration of MCP‐1 and TNF‐α. Accordingly, BPS suppressed the LPS‐stimulated Mcp‐1 and Tnf‐α mRNA expression in cultured Kupffer cells prepared from WT mice. PGI2‐IP signaling plays a crucial role in the development and progression of steatohepatitis by modulating the inflammatory response, leading to augmented oxidative stress. We suggest that the PGI2‐IP system is an attractive therapeutic target for treating patients with NASH.—Kumei, S., Yuhki, K‐I., Kojima, F., Kashiwagi, H, Imamichi, Y., Okumura, T., Narumiya, S., Ushikubi, F. Prostaglandin I2 suppresses the development of diet‐induced nonalcoholic steatohepatitis in mice. FASEB J. 32, 2354–2365 (2018). www.fasebj.org

Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity

The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A 2 receptor agonist, and that induced by collagen with respective IC 50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid–induced TXA 2 production in murine platelets with an IC 50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA 2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I 2 receptor and PGE 2 receptor subtypes EP 2 and EP 4 , were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA 2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC 50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA 2 production in platelets, leading to inhibition of platelet aggregation.