Association of postoperative atrial fibrillation with higher dosing ratios of protamine-to-heparin.

Background: Postoperative atrial fibrillation (POAF) is defined as new-onset AF in the immediate postoperative period. The relatively high incidence of POAF after cardiac surgery is well described, but pathophysiological mechanisms underlying the initiation, maintenance, and progression of POAF may be multifactorial and have not yet been comprehensively characterized. One of the mechanisms includes altered Ca2+ kinetics. Accumulating evidence has suggested that altered atrial cytosolic calcium handling contributes to the development of POAF, protamine reversibly modulates the calcium release channel/ryanodine receptor 2 (RyR2) and voltage-dependent cardiac RyR2. However, it is currently unknown whether such abnormalities contribute to the arrhythmogenic substrate predisposing patients to the development of POAF. Methods: We have retrospectively analyzed 147 patients who underwent cardiac surgery with cardiopulmonary bypass support. Of these, 40 patients were excluded from the analysis because of pre-existing AF. All patients received heparin followed by protamine at different dosing ratios of protamine-to-heparin, depending on the periods studied. Results: The dosing ratio of protamine-to-heparin = 1.0 was compared with higher dosing ratios of protamine-to-heparin >1.0 up to 1.7. POAF developed in 15 patients (15/107 = 14%), of these, 5 out of 57 patients (33.3%) in the dosing ratio of protamine-to-heparin = 1.0 and 10 out of 35 patients (66.7%) in the higher dosing ratios of protamine-to-heparin. Statistical significance was observed in patients with higher dosing ratios of protamine-to-heparin, compared with the dosing ratio of protamine-to-heparin = 1.0 (odds ratio = 3.890, 95% CI = 1.130-13.300, p-value = 0.031). When types of diseases were analyzed in terms of higher dosing ratios of protamine-to-heparin, only valvular disorders were significantly associated with POAF (p = 0.04). Conclusions: Protamine is clinically utilized to reverse heparin overdose and has been shown to display immunological and inflammatory alterations. However, its association with POAF has not been reported. Our results provide evidence that higher dosing ratios of protamine-to-heparin may increase the incidence of POAF.

Arteriosclerosis Derived from Cutaneous Inflammation Is Ameliorated by the Deletion of IL-17A and IL-17F.

The skin is one of the major immune organs producing large amounts of proinflammatory and inflammatory cytokines in response to internal or exogenous stimuli, inducing systemic inflammation in various internal organs. In recent years, organ damage associated with inflammatory skin diseases such as psoriasis and atopic dermatitis has received increasing attention, and vascular disorder such as arteriosclerosis is one of the serious complications of chronic inflammatory skin diseases. However, the detailed mechanism of arteriosclerosis in dermatitis and the role of cytokines have not been clarified so far. In the current study, using a spontaneous dermatitis model, we investigated the pathophysiology of arteriosclerosis and the treatment option for inflammatory skin conditions. We employed spontaneous dermatitis model mice overexpressing human caspase-1 in the epidermal keratinocyte (Kcasp1Tg). The thoracic and abdominal aorta was investigated histologically. GeneChip and RT-PCR analysis were performed to measure the changes in mRNA levels in the aorta. To elucidate the direct effect on the artery by major inflammatory cytokines, endothelial cells, vascular smooth muscle cells, and fibroblast cells were co-cultured with several cytokines, and mRNA expression levels were measured. In order to observe the efficacy of IL-17A/F in arteriosclerosis, cross-mating with IL-17A, IL-17F, and IL-17A/F deficient mice was performed. Finally, we also measured snap tension in the abdominal aorta in WT, Kcasp1Tg, and IL17A/F-deficient mice. Kcasp1Tg showed a decrease in the diameter of the abdominal aorta compared to wild-type mice. mRNA levels for six genes including Apol11b, Camp, Chil3, S100a8, S100a9, and Spta1 were increased in the abdominal aorta of Kcasp1Tg. Some of the above mRNA levels were also increased in the co-culture with major inflammatory cytokines, IL-17A/F, IL-1ß, and TNF-α. Dermatitis improved and mRNA levels were partially ameliorated in Kcasp1Tg with IL-17A/F deletion. Arterial fragility was also evidenced in the inflammatory model, but arterial flexibility was revealed in the IL-17A/F deletion model. Severe dermatitis is closely related to secondary arteriosclerosis caused by the persistent release of inflammatory cytokines. The results also proved that treatment against IL-17A and F may ameliorate arteriosclerosis.

Perinatal hypoxia aggravates occlusive pulmonary vasculopathy in SU5416/hypoxia-treated rats later in life.

Pulmonary arterial hypertension (PAH) is a fatal disease, which is characterized by occlusive pulmonary vascular disease (PVD) in small pulmonary arteries. It remains unknown whether perinatal insults aggravate occlusive PVD later in life. We tested the hypothesis that perinatal hypoxia aggravates PVD and survival in rats. PVD was induced in rats with/without perinatal hypoxia (embryonic day 14 to postnatal day 3) by injecting SU5416 at 7 wk of age and subsequent exposure to hypoxia for 3 wk (SU5416/hypoxia). Hemodynamic and morphological analyses were performed in rats with/without perinatal hypoxia at 7 wk of age (baseline rats, n = 12) and at 15 wk of age in 4 groups of rats: SU5416/hypoxia or control rats with/without perinatal hypoxia (n = 40). Pulmonary artery smooth muscle cells (PASMCs) from the baseline rats with/without perinatal hypoxia were used to assess cell proliferation, inflammation, and genomic DNA methylation profile. Although perinatal hypoxia alone did not affect survival, physiological, or pathological parameters at baseline or at the end of the experimental period in controls, perinatal hypoxia decreased weight gain and survival rate and increased right ventricular systolic pressure, right ventricular hypertrophy, and indices of PVD in SU5416/hypoxia rats. Perinatal hypoxia alone accelerated the proliferation and inflammation of cultured PASMCs from baseline rats, which was associated with DNA methylation. In conclusion, we established the first fatal animal model of PAH with worsening hemodynamics and occlusive PVD elicited by perinatal hypoxia, which was associated with hyperproliferative, proinflammatory, and epigenetic changes in cultured PASMCs. These findings provide insights into the treatment and prevention of occlusive PVD.

CRISPR-mediated Bmpr2 point mutation exacerbates late pulmonary vasculopathy and reduces survival in rats with experimental pulmonary hypertension.

BACKGROUND: Patients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (Bmpr2) mutations present earlier with severe hemodynamic compromise and have poorer survival outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with Bmpr2 mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant Bmpr2 mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats. METHODS: A monoallelic single nucleotide insertion in exon 1 of Bmpr2 (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed. RESULTS: The +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/[LV + S]) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type. CONCLUSIONS: The present study demonstrates that the Bmpr2 mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.

Lung damage created by high tidal volume ventilation in rats with monocrotaline-induced pulmonary hypertension.

BACKGROUND: Rats with chronic hypoxia-induced non-inflammatory pulmonary hypertension (PH) are resistant to ventilator-induced lung injury. We investigated the effect of high tidal volume ventilation in another model of PH, monocrotaline (MCT)-induced PH, which is a type of inflammatory PH. METHODS: PH was induced in rats by subcutaneous injection with 60 mg/kg MCT. Normal control rats, rats at 2 weeks after MCT injection (MCT2), and rats at 3 weeks after MCT injection (MCT3) were ventilated with low tidal volume (LV, 6 mL/kg) or high tidal volume (HV, 35 mL/kg) for 2 h with room air without positive end-expiratory pressure. Arterial oxygen pressure (PaO2) and Evans blue dye (EBD) extravasation were measured. Hypertensive pulmonary vascular remodeling was assessed morphometrically by the percentage of muscularized peripheral pulmonary arteries (%Muscularization) and the media wall thickness to external diameter ratio, namely percentage medial wall thickness (%MWT). To assess inflammation, lung IκB protein and cytokine mRNA expression levels were assessed. RESULTS: Baseline mean pulmonary arterial pressure was significantly higher in MCT rats (normal, 15.4 ± 0.5 mmHg; MCT2, 23.7 ± 0.9; and MCT3, 34.5 ± 1.5). After 2-h ventilation, PaO2 was significantly lower in the HV groups compared with the LV groups in normal and MCT2 rats, but not in MCT3 rats. Impairment of oxygenation with HV was less in MCT3 rats compared with normal and MCT2 rats. Among the HV groups, MCT3 rats showed significantly lower levels of EBD extravasation than normal and MCT2 rats. HV significantly downregulated IκB protein expression in normal and MCT3 rats and increased IL-6, MCP-1, CXCL-1 (MIP-1), and IL-10 mRNA levels in MCT3 rats. %Muscularization, %MWT, and the expression of lung elastin were significantly higher in MCT3 rats than in normal and MCT2 rats. CONCLUSION: We found that HV-associated damage might be reduced in MCT-induced PH rats compared with normal rats. The results of this and earlier studies suggest that hypertensive pulmonary vascular structural changes might be protective against the occurrence of ventilator-induced lung injury, irrespective of the etiology of PH.

Model difference in the effect of cilostazol on the development of experimental pulmonary hypertension in rats.

BACKGROUND: Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. METHODS: Fifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. The mean pulmonary artery pressure (mPAP), the right ventricle weight-to-left ventricle + septum weight ratio (RV/LV + S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Levels of the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB proteins in lung tissue were measured using Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. RESULTS: mPAP [35.1 ± 1.7 mmHg (MCT) (n = 9) vs. 16.6 ± 0.7 (control) (n = 9) (P < 0.05); 29.1 ± 1.5 mmHg (CH) (n = 10) vs. 17.5 ± 0.5 (control) (n = 10) (P < 0.05)], RV/LV + S [0.40 ± 0.01 (MCT) (n = 18) vs. 0.24 ± 0.01 (control) (n = 10) (P < 0.05); 0.41 ± 0.03 (CH) (n = 13) vs. 0.27 ± 0.06 (control) (n = 10) (P < 0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1 ± 1.1 mmHg (n = 11) (P < 0.05), RV/LV + S 0.30 ± 0.01 (n = 14) (P < 0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and increased in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats. CONCLUSIONS: We found model differences in the effect of CLZ on PH development. CLZ might exert a preventive effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might depend on the PH etiology.

Use of Augmented Reality to Assist Teaching for Future Perfusionists in Extracorporeal Technology.

The aim of this study was to foster the better perfusion education when providing extracorporeal circulation (ECC) technology for future perfusionists. For this purpose, we have developed an augmented reality (AR) program for ECC students. Currently, the cost of equipment and its simulator is high. Furthermore, it is desirable for ECC students to practice at any time. AR describes user experiences that add 2D (plane detection) or 3D elements to the live view from a device's camera in a way that makes those elements appear to inhabit the real world. We can use these technologies to create AR experiences using the back camera of a smartphone or tablet. We can also build our own instrument with custom visualization and data analysis. Although AR technology may not be new, its potential in ECC student education is just beginning to be explored. Unlike other computing technologies, AR interfaces offer seamless interaction between the real and virtual worlds, a tangible interface metaphor, and a means for transitioning between real and virtual worlds. Here, we have shown our experiences of cost-effective AR technology for future perfusionists.

Acute undifferentiated leukemia (stem cell acute leukemia) showing differentiation to Langerhans cell-like cells in lymph nodes.

A 75-year-old woman was referred to our hospital with suspected leukemia. Complete blood count demonstrated WBC 3,810/µl with 26% blasts, Hb of 11.7 g/dl and Plt of 18.0×104/µl. Bone marrow aspiration revealed blasts (86.3%) with expressions of CD34, CD7, TdT, CD33, and CD117. MPO was negative. Chromosomal analysis of the bone marrow showed isolated trisomy 10 in all leukemic cells (20/20). Swelling of superficial lymph nodes was also observed. Cervical lymph node biopsy revealed leukemic blasts which had the same phenotype as those in the bone marrow except that proliferation of Langerhans cell-like cells (LCs) was observed in the paracortex. LCs had pale cytoplasm and grooved nuclei, and were positive for both CD1a and S100 protein. Trisomy 10 was detected in both the leukemic blasts and the LCs by fluorescence in situ hybridization. This rare case strongly suggests leukemic cells to differentiate into LCs.

Sarpogrelate hydrochloride, a serotonin 5HT2A receptor antagonist, ameliorates the development of chronic hypoxic pulmonary hypertension in rats.

PURPOSE: The purpose of the present study was to determine if sarpogrelate hydrochloride (SPG), a serotonin 5HT2A receptor antagonist, prevented the development of chronic hypoxia-induced pulmonary hypertension (PH) and hypertensive pulmonary vascular remodeling. METHODS: Forty-one male Sprague-Dawley rats were exposed to hypobaric hypoxia (380 mmHg, 10 % oxygen) or room air and administered 50 mg/kg SPG or vehicle by gavage once daily from day -2 to day 14. The mean pulmonary artery pressure (PAP) and right ventricular hypertrophy (RVH) were measured. Hypertensive pulmonary vascular remodelings were assessed morphometrically by light microscopy. Serotonin-induced contraction was determined in isolated pulmonary artery rings from 24 rats. In another set of rats, Western blotting, real-time polymerase chain reaction and immunofluorescent staining (n = 9) for lung tissue were performed. RESULTS: Chronic hypoxia induced a rise in mean PAP and RVH, increased the percentage of muscularized arteries in peripheral pulmonary arteries and medial wall thickness in small muscular arteries, and potentiated serotonin-induced contraction, each of which was significantly (p < 0.05) ameliorated by SPG. Chronic hypoxia significantly increased the expression of endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) protein levels, cyclic guanosine monophosphate, and matrix metalloproteinase-13 (MMP-13) mRNA levels in whole lung tissues. SPG increased peNOS expression in the immunofluorescent staining of peripheral pulmonary arteries from chronic hypoxic rats and decreased the MMP-13 mRNA in lung tissue in chronic hypoxic rats. CONCLUSIONS: The administration of SPG ameliorated the development of chronic hypoxic PH and hypertensive pulmonary vascular changes.

Effect of thrombomodulin on the development of monocrotaline-induced pulmonary hypertension.

PURPOSE: The purpose of the present study was to investigate whether thrombomodulin (TM) prevents the development of pulmonary hypertension (PH) in monocrotaline (MCT)-injected rats. METHODS: Human recombinant TM (3 mg/kg/2 days) or saline were given to MCT-injected male Sprague-Dawley rats for 19 (n = 14) or 29 (n = 11) days. Control rats (n = 6) were run for 19 days. The mean pulmonary artery pressure (mPAP), right ventricular hypertrophy (RVH), percentages of muscularized peripheral arteries (%muscularization), and medial wall thickness of small muscular arteries (%MWT) were measured. To determine inflammatory and coagulation responses, broncho-alveolar lavage fluid (BALF) was analyzed in another set of rats (n = 29). Western blotting for endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (peNOS) in the lung tissue was performed in separate rats (n = 13). Survival was determined in 60 rats. RESULTS: MCT increased mPAP, RVH, %muscularization, and %MWT. TM treatment significantly reduced mPAP, %muscularization, and %MWT in peripheral arteries with an external diameter of 50-100 µm in 19 days after MCT injection, but the effect was lost after 29 days. MCT increased the levels of tumor necrosis factor alpha, monocyte chemoattractant protein-1, and thrombin-antithrombin complex in BALF. Expression of eNOS increased in MCT rats, while peNOS decreased. The relative amount of peNOS to total eNOS increased in MCT/TM rats compared to MCT/Vehicle rats. A Kaplan-Meier survival curve showed no difference with and without TM. CONCLUSION: Although the administration of TM might slightly delay the progression of MCT-induced PH, the physiological significance for treatment is limited, since the survival rate was not improved.

Skin ulcer is a predictive and prognostic factor of acute or subacute interstitial lung disease in dermatomyositis.

The aim of this study was to determine whether skin ulcer can be used as a predictive and prognostic factor of acute/subacute interstitial lung disease (ILD) in Japanese patients with dermatomyositis (DM). We reviewed the medical records of 39 consecutive DM patients who were admitted to Tokyo Metropolitan Komagome Hospital from January 2000 to December 2009. The mean follow-up period was 63.9 ± 51.6 months. Fifteen patients had acute/subacute ILD and 11 patients had chronic ILD. Seven out of 15 acute/subacute ILD led to respiratory failure and 3 of them died due to ILD. Skin ulcers were observed in 5 out of 15 patients with acute/subacute ILD (33.3 %) and in 2 out of 24 patients without acute/subacute ILD (8.3 %). The presence of skin ulcers was revealed to be a significant predictive factor for acute/subacute ILD among various parameters by multivariate analysis. In the 15 patients with acute/subacute ILD, the presence of skin ulcers was a significant poor prognostic factor (p = 0.0231) and the cumulative survival rate of patients with skin ulcers was 53.3 % for 12 months. Skin ulcer is a significant predictive and prognostic factor of acute/subacute ILD in patients with DM.

Clinical and pathological features of Churg Strauss syndrome among a Japanese population: a case series of 18 patients.

BACKGROUND: Many autoimmune diseases differ in individual of different races, but there has been very scarce information on the clinicopathological features of Churg Strauss syndrome (CSS) among Asians patients. OBJECTIVE: To clarify the clinicopathological details of Japanese CSS patients. METHODS: The medical records of CSS patients hospitalized in 1980-2007 were carefully reviewed. RESULTS: Seventeen patients fulfilled the Japanese Ministry of Health, Labour and Welfare (MHLW) criteria and all 18 fulfilled the American College of Rheumatology criteria. Sixteen patients (89%) had the history of asthma. Frequently involved organs were peripheral nerves (PNS) (94%), skin (50%), gastrointestinal tract (33%), kidney (22%), and heart (17%). The mean (range) eosinophil count, C-reactive protein, and number of damaged organs was 18,108 (3,820-36,760)/microL, 51 (0-126) mg/L, and 2.7 (1-6), respectively. Four patients died, of whom three had heart involvement while only one without it died (100 versus 9%, respectively, p = 0.0088). Regarding the pathology, vasculitis was observed in six of seven skin but in only 2 of 10 PNS biopsies. Eosinophilia was found in all of the tissues except for PNS and muscle (40%). Granuloma was observed in only three of the total of 29 biopsies. CONCLUSION: The usefulness of MHLW criteria was verified. The clinical features of Japanese CSS patients were mostly similar to those previously reported, except for lower asthma- and ANCA-positivity rates. Regarding the pathology, vasculitis and eosinophilia were much more frequently observed in skin.

The Isoquinoline-Sulfonamide Compound H-1337 Attenuates SU5416/Hypoxia-Induced Pulmonary Arterial Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and right heart failure. Selective pulmonary vasodilators have improved the prognosis of PAH; however, they are not able to reverse pulmonary vascular remodeling. Therefore, a search for new treatment agents is required. H-1337 is an isoquinoline-sulfonamide compound that inhibits multiple serine/threonine kinases, including Rho-associated protein kinase (ROCK) and mammalian target of rapamycin (mTOR). Here, we investigated the effects of H-1337 on pulmonary hypertension and remodeling in the pulmonary vasculature and right ventricle in experimental PAH induced by SU5416 and hypoxia exposure. H-1337 and H-1337M1 exerted inhibitory effects on ROCK and Akt. H-1337 inhibited the phosphorylation of myosin light chain and mTOR and suppressed the proliferation of smooth muscle cells in vitro. H-1337 treatment also suppressed the phosphorylation of myosin light chain and mTOR in the pulmonary vasculature and decreased right ventricular systolic pressure and the extent of occlusive pulmonary vascular lesions. Furthermore, H-1337 suppressed aggravation of right ventricle hypertrophy. In conclusion, our data demonstrated that inhibition of ROCK and mTOR pathways with H-1337 suppressed the progression of pulmonary vascular remodeling, pulmonary hypertension, and right ventricular remodeling.

Effect of all-trans-retinoic acid on the development of chronic hypoxia-induced pulmonary hypertension.

BACKGROUND: An earlier study showed that all-trans-retinoic acid (ATRA) prevents the development of monocrotalin-induced pulmonary hypertension (PH). The purpose of the present study was to determine the effect of ATRA on another model of chronic hypoxia-induced PH. METHODS AND RESULTS: Male Sprague-Dawley rats were given 30 mg/kg ATRA or vehicle only by gavage once daily for 14 days during hypobaric hypoxic exposure. Chronic hypoxic exposure induced PH, right ventricular hypertrophy (RVH), and hypertensive pulmonary vascular changes. Quantitative morphometry of the pulmonary arteries showed that ATRA treatment significantly reduced the percentage of muscularized arteries in peripheral pulmonary arteries only with an external diameter between 15 and 50 microm. ATRA treatment also significantly reduced the medial wall thickness in small muscular arteries only with an external diameter between 50 and 100 microm. Unfortunately, these reductions did not accompany the lowering of pulmonary artery pressure nor decrease in RVH. Chronic hypoxia-induced PH rats with ATRA had a loss in body weight. Chronic hypoxia increased the expression of endothelial nitric oxide synthase in the lung on western blotting and immunohistochemistry, in which ATRA treatment had no effect. CONCLUSIONS: The administration of ATRA might not have a therapeutic role in preventing the development of chronic hypoxia-induced PH, because of body weight loss and the subtle preventable effects of vascular changes.

Colforsin-induced vasodilation in chronic hypoxic pulmonary hypertension in rats.

PURPOSE: Colforsin, a water-soluble forskolin derivative, directly activates adenylate cyclase and thereby increases the 3',5'-cyclic adenosine monophosphate (cAMP) level in vascular smooth muscle cells. In this study, we investigated the vasodilatory action of colforsin on structurally remodeled pulmonary arteries from rats with pulmonary hypertension (PH). METHODS: A total of 32 rats were subjected to hypobaric hypoxia (380 mmHg, 10% oxygen) for 10 days to induce chronic hypoxic PH, while 39 rats were kept in room air. Changes in isometric force were recorded in endothelium-intact (+E) and -denuded (-E) pulmonary arteries from the PH and control (non-PH) rats. RESULTS: Colforsin-induced vasodilation was impaired in both +E and -E arteries from PH rats compared with their respective controls. Endothelial removal did not influence colforsin-induced vasodilation in the arteries from control rats, but attenuated it in arteries from PH rats. The inhibition of nitric oxide (NO) synthase did not influence colforsin-induced vasodilation in +E arteries from controls, but attenuated it in +E arteries from PH rats, shifting its concentration-response curve closer to that of -E arteries from PH rats. Vasodilation induced by 8-bromo-cAMP (a cell-permeable cAMP analog) was also impaired in -E arteries from PH rats, but not in +E arteries from PH rats, compared with their respective controls. CONCLUSIONS: cAMP-mediated vasodilatory responses without beta-adrenergic receptor activation are impaired in structurally remodeled pulmonary arteries from PH rats. In these arteries, endothelial cells presumably play a compensatory role against the impaired cAMP-mediated vasodilatory response by releasing NO (and thereby attenuating the impairment). The results suggest that colforsin could be effective in the treatment of PH.

A non-selective endothelin receptor antagonist bosentan modulates kinetics of bone marrow-derived cells in ameliorating pulmonary hypertension in mice.

The aim of this study was to investigate whether a dual endothelin receptor antagonist bosentan modulates the kinetics of bone marrow-derived stem cells in inhibiting the development of pulmonary hypertension. Bone marrow chimeric mice, transplanted with enhanced green fluorescent protein (eGFP)-positive bone marrow mononuclear cells, were exposed to hypobaric hypoxia or kept in the ambient air, and were daily treated with bosentan sodium salt or saline for 21 days. After the treatment period, right ventricular pressure was measured and pulmonary vascular morphometry was conducted. Incorporation of bone marrow-derived cells was analyzed by immunohistochemistry. Gene expression and protein level in the lung tissue were evaluated by quantitative real-time PCR and western blotting, respectively. The results showed that, in hypoxic mice, right ventricular pressure and the percentage of muscularized vessel were increased and pulmonary vascular density was decreased, each of which was reversed by bosentan. Bone marrow-derived endothelial cells and macrophages in lungs were increased by hypoxia. Bosentan promoted bone marrow-derived endothelial cell incorporation but inhibited macrophage infiltration into lungs. Quantitative real-time PCR analysis revealed that interleukin 6, stromal cell-derived factor-1, and monocyte chemoattractant protein-1 were upregulated by hypoxia, in which interleukin 6 and monocyte chemoattractant protein-1 were downregulated and stromal cell-derived factor-1 was upregulated by bosentan. Protein level of endothelial nitric oxide synthase (eNOS) in the whole lung was significantly upregulated by hypoxia, which was further upregulated by bosentan. Bosentan modulated kinetics of bone marrow-derived ECs and macrophages and related gene expression in lungs in ameliorating pulmonary hypertension in mice. Altered kinetics of bone marrow-derived stem cells may be a novel mechanism of the endothelin receptor blockade in vivo and confer a new understanding of the therapeutic basis for pulmonary hypertension.

The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension.

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.

Role of thrombin in interleukin-5 expression from basophils.

Interleukin-5 (IL-5) plays a key role in the pathogenesis of bronchial asthma. Thrombin is a procoagulant factor that has been also reported to participate in the inflammatory response by stimulating the secretion of cytokines. Interaction of inflammatory cells with airway epithelial cells may also promote the secretion of cytokines. However, the role of thrombin and cell-to-cell interaction in pathogenesis of allergic inflammation is unclear. In this study, we evaluated the role of thrombin and cell-to-cell interaction in the secretion of IL-5 from basophils. The human basophil cell line KU-812 was used in the assays. Thrombin and co-culture with alveolar epithelial cells significantly stimulated the secretion of IL-5 from KU-812 cells as compared to controls. Secretion of IL-5 was synergistically stimulated when KU-812 cells were incubated in the presence of both thrombin and alveolar epithelial cells. Co-culture of KU-812 cells with epithelial cells significantly increased the expression of tissue factor, an activator of coagulation activation, in a cell dose-dependent manner. Secretion of IL-5 from KU-812 basophils co-cultured with epithelial cells was significantly inhibited by LY294002, an inhibitor of phosphatidylinositol 3-kinase. These results suggest that thrombin and cell interaction with lung epithelial cells may augment the inflammatory response in allergic diseases by stimulating the secretion of IL-5 from basophils.

Tenascin-C synthesized in both donor grafts and recipients accelerates artery graft stenosis.

OBJECTIVE: Tenascin-C, an extracellular matrix glycoprotein, is thought to play an important role in neointimal hyperplasia of artery bypass grafts. In this study, the direct contribution of tenascin-C to neointimal hyperplasia of free artery grafts and the origin of tenascin-C-producing cells were examined using tenascin-C transgenic mice. METHODS AND RESULTS: Abdominal aorta-to-carotid artery interposition grafting was performed in mice. When grafts from wild-type mice were transplanted to wild-type, neointimal hyperplasia was observed in the grafts at days 14 and 28. Immunohistochemical staining showed strong expression of tenascin-C in the media and neointima of the grafts. Much less neointimal hyperplasia was seen when grafts from tenascin-C-deficient mice were transplanted to tenascin-C-deficient mice. In tenascin-C-deficient grafts transplanted to wild-type mice, tenascin-C deposition was observed only in the neointima. In the reverse combination, deposition was seen in the media and neointima. The source of the tenascin-C-producing cells was analyzed using heterozygous mice that identically express both tenascin-C and LacZ. While LacZ-positive cells were seen only in the neointima of artery grafts from wild-type transplanted to mutant mice, positive cells were detected in both the neointima and media in grafts from mutant to wild-type mice. CONCLUSIONS: We presented direct evidence that tenascin-C is a crucial molecule in neointimal hyperplasia in free artery grafts, and that tenascin-C-producing cells are derived from both donor grafts and recipients.