Takayuki Fujino

Thromboxane A2 and prostaglandin F2α mediate inflammatory tachycardia

Systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. Prostanoids, including prostaglandin (PG) D2, PGE2, PGF2α, PGI2 and thromboxane (TX) A2, exert their actions through specific receptors: DP, EP (EP1, EP2, EP3, EP4), FP, IP and TP, respectively. Here we have examined the roles of prostanoids in inflammatory tachycardia using mice that lack each of these receptors individually. The TXA2 analog I-BOP and PGF2α each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The β-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Our results show that inflammatory tachycardia is caused by a direct action on the heart of TXA2 and PGF2α formed under systemic inflammatory conditions.

Prostaglandin E2 protects the heart from ischemia-reperfusion injury via its receptor subtype EP4.

Background—In the heart with acute myocardial infarction, production of prostaglandin (PG) E2 increases significantly. In addition, several subtypes of PGE2 receptors (EPs) have been reported to be expressed in the heart. The role of PGE2 in cardiac ischemia-reperfusion (I/R) injury, however, remains unknown. We intended to clarify the role of PGE2 via EP4, an EP subtype, in I/R injury using mice lacking EP4 (EP4−/− mice). Methods and Results—In murine cardiac ventricle, competitive reverse transcription–polymerase chain reaction revealed the highest expression level of EP4 mRNA among EP mRNAs. EP4−/− mice had larger infarct size than wild-type mice in a model of I/R; the left anterior descending coronary artery was occluded for 1 hour, followed by 24 hours of reperfusion. In addition, isolated EP4−/− hearts perfused according to the Langendorff technique had greater functional and biochemical derangements in response to I/R than wild-type hearts. In vitro, AE1-329, an EP4 agonist, raised cAMP concentration remarkably in noncardiomyocytes, whereas the action was weak in cardiomyocytes. When 4819-CD, another EP4 agonist, was administered 1 hour before coronary occlusion, it reduced infarct size significantly in wild-type mice. Notably, a similar cardioprotective effect was observed even when it was administered 50 minutes after coronary occlusion. Conclusions—Both endogenous PGE2 and an exogenous EP4 agonist protect the heart from I/R injury via EP4. The potent cardioprotective effects of 4819-CD suggest that the compound would be useful for treatment of acute myocardial infarction.

Decreased susceptibility to renovascular hypertension in mice lacking the prostaglandin I2 receptor IP

Persistent reduction of renal perfusion pressure induces renovascular hypertension by activating the renin-angiotensin-aldosterone system; however, the sensing mechanism remains elusive. Here we investigated the role of PGI2 in renovascular hypertension in vivo, employing mice lacking the PGI2 receptor (IP-/- mice). In WT mice with a two-kidney, one-clip model of renovascular hypertension, the BP was significantly elevated. The increase in BP in IP-/- mice, however, was significantly lower than that in WT mice. Similarly, the increases in plasma renin activity, renal renin mRNA, and plasma aldosterone in response to renal artery stenosis were all significantly lower in IP-/- mice than in WT mice. All these parameters were measured in mice lacking the four PGE2 receptor subtypes individually, and we found that these mice had similar responses to WT mice. PGI2 is produced by COX-2 and a selective inhibitor of this enzyme, SC-58125, also significantly reduced the increases in plasma renin activity and renin mRNA expression in WT mice with renal artery stenosis, but these effects were absent in IP-/- mice. When the renin-angiotensin-aldosterone system was activated by salt depletion, SC-58125 blunted the response in WT mice but not in IP-/- mice. These results indicate that PGI2 derived from COX-2 plays a critical role in regulating the release of renin and consequently renovascular hypertension in vivo.

Augmented Cardiac Hypertrophy in Response to Pressure Overload in Mice Lacking the Prostaglandin I2 Receptor

Background—In the heart, the expressions of several types of prostanoid receptors have been reported. However, their roles in cardiac hypertrophy in vivo remain unknown. We intended to clarify the roles of these receptors in pressure overload–induced cardiac hypertrophy using mice lacking each of their receptors. Methods and Results—We used a model of pressure overload–induced cardiac hypertrophy produced by banding of the transverse aorta in female mice. In wild-type mice subjected to the banding, cardiac hypertrophy developed during the observation period of 8 weeks. In mice lacking the prostaglandin (PG) I2 receptor (IP−/−), however, cardiac hypertrophy and cardiomyocyte hypertrophy were significantly greater than in wild-type mice at 2 and 4 weeks but not at 8 weeks, whereas there was no such augmentation in mice lacking the prostanoid receptors other than IP. In addition, cardiac fibrosis observed in wild-type hearts was augmented in IP−/− hearts, which persisted for up to 8 weeks. In IP−/− hearts, the expression level of mRNA for atrial natriuretic peptide, a representative marker of cardiac hypertrophy, was significantly higher than in wild-type hearts. In vitro, cicaprost, an IP agonist, reduced platelet-derived growth factor–induced proliferation of wild-type noncardiomyocytes, although it could not inhibit cardiotrophin-1–induced hypertrophy of cardiomyocytes. Accordingly, cicaprost increased cAMP concentration efficiently in noncardiomyocytes. Conclusions—IP plays a suppressive role in the development of pressure overload–induced cardiac hypertrophy via the inhibition of both cardiomyocyte hypertrophy and cardiac fibrosis. Both effects have been suggested as originating from the action on noncardiomyocytes rather than cardiomyocytes.

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.

Prostaglandin I2 promotes recruitment of endothelial progenitor cells and limits vascular remodeling

Objective—Endothelial progenitor cells (EPCs) play an important role in the self-healing of a vascular injury by participating in the reendothelialization that limits vascular remodeling. We evaluated whether prostaglandin I2 plays a role in the regulation of the function of EPCs to limit vascular remodeling. Methods and Results—EPCs (Lin−cKit+Flk-1+ cells) were isolated from the bone marrow (BM) of wild-type (WT) mice or mice lacking the prostaglandin I2 receptor IP (IP−/− mice). Reverse transcription–polymerase chain reaction analysis showed that EPCs among BM cells specifically express IP. The cellular properties of EPCs, adhesion, migration, and proliferation on fibronectin were significantly attenuated in IP-deficient EPCs compared with WT EPCs. In contrast, IP agonists facilitated these functions in WT EPCs, but not in IP-deficient EPCs. The specific deletion of IP in BM cells, which was performed by transplanting BM cells of IP−/− mice to WT mice, accelerated wire injury–mediated neointimal hyperplasia in the femoral artery. Notably, transfused WT EPCs, but not IP-deficient EPCs, were recruited to the injured vessels, participated in reendothelialization, and efficiently rescued the accelerated vascular remodeling. Conclusion—These findings clearly indicate that the prostaglandin I2-IP system is essential for EPCs to accomplish their function and plays a critical role in the regulation of vascular remodeling.

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.

An Oral Adsorbent, AST-120, Suppresses Oxidative Stress in Uremic Rats

Background: The production of reactive oxygen species (ROS) has been suggested to play an important role in the progression of chronic kidney disease (CKD). An oral adsorbent, AST-120, removes uremic toxins such as indoxyl sulfate (IS) and delays the progression of CKD, but the effect on ROS production is unknown. The present study aimed to determine whether AST-120 reduces oxidative stress in uremic rat kidneys using markers of ROS production such as acrolein and 8-hydroxy-2′-deoxyguanosine (8-OHdG). Methods: Daily administration of AST-120 was started 6 weeks after 5/6 nephrectomy and continued for 18 weeks. The changes in metabolic data, serum and urine IS levels, urinary excretion of markers of oxidative stress, and renal histological findings were investigated in uremic rats with or without AST-120 treatment. Results: In parallel with the increase in serum and urine IS, the serum creatinine, urinary protein and acrolein levels started to increase at 6 weeks, but urinary 8-OHdG remained unchanged and significantly increased at 18 weeks in uremic rats. AST-120 markedly and significantly attenuated increases in uremic toxins and oxidative stress levels as well as the histological changes in glomerular sclerosis, interstitial fibrosis, and the tubular staining of 8-OHdG. Conclusion: AST-120 suppressed the progression of CKD, at least in part, via attenuation of oxidative stress induced by uremic toxin.

Thromboxane A2 Regulates Vascular Tone via Its Inhibitory Effect on the Expression of Inducible Nitric Oxide Synthase

Background—Circulatory failure in sepsis arises from vascular hyporesponsiveness, in which nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. Details of the cross talk between thromboxane (TX) A2 and the iNOS–NO system, however, remain unknown. We intended to clarify the role of TXA2, via the cross talk, in vascular hyporesponsiveness. Methods and Results—We examined cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the TXA2 receptor (TP−/− mice). The cytokine-induced iNOS expression and NO production observed in wild-type VSMCs were significantly augmented in TP−/− VSMCs, indicating an inhibitory effect of endogenous TXA2 on iNOS expression. Furthermore, in indomethacin-treated wild-type VSMCs, U-46619, a TP agonist, inhibited cytokine-induced iNOS expression and NO production in a concentration-dependent manner, effects absent from TP−/− VSMCs. In an ex vivo system, the cytokine-induced hyporesponsiveness of aortas to phenylephrine was significantly augmented in TP−/− aorta but was almost completely canceled by aminoguanidine, an iNOS inhibitor. Accordingly, cytokine-induced NO production was significantly higher in TP−/− aorta than in wild-type aorta. Moreover, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo only in wild-type mice. Conclusions—These results suggest that TXA2 has a protective role against the development of vascular hyporesponsiveness via its inhibitory action on the iNOS–NO system under pathological conditions such as sepsis.

Roles of calcineurin and calcium/calmodulin-dependent protein kinase II in pressure overload-induced cardiac hypertrophy

Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiac hypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload without the influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, we examined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, and protein synthesis using heart perfusion model. The hearts isolated from Sprague-Dawley rats were perfused according to the Langendorff technique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated by its complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by its autophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fos expression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fos expression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failed to affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-induced acceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.