Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR.

Recently, we confirmed that in human aortic valve interstitial cells (HAVICs) isolated from patients with aortic valve stenosis (AVS), calcification is induced in high inorganic phosphate (high-Pi) medium by warfarin (WFN). Because WFN is known as a vitamin K antagonist, reducing the formation of blood clots by vitamin K cycle, we hypothesized that vitamin K regulates WFN-induced HAVIC calcification. Here, we sought to determine whether WFN-induced HAVIC calcification in high-Pi medium is inhibited by menaquinone-4 (MK-4), the most common form of vitamin K2 in animals. HAVICs obtained from patients with AVS were cultured in α-modified Eagle's medium containing 10% FBS, and when the cells reached 80%-90% confluency, they were further cultured in the presence or absence of MK-4 and WFN for 7 days in high-Pi medium (3.2 mM Pi). Intriguingly, in high-Pi medium, MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated the calcification when used alone (at 10 nM). Furthermore, MK-4 enhanced alkaline phosphatase (ALP) activity in HAVICs, and 7 days of MK-4 treatment markedly upregulated the gene expression of the calcification marker bone morphogenetic protein 2 (BMP2). Notably, MK-4-induced calcification was potently suppressed by two pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol; conversely, PXR activity was weakly increased, but in a statistically significant and dose-dependent manner, by MK-4. Lastly, in physiologic-Pi medium, MK-4 increased BMP2 gene expression and accelerated excess BMP2 (30 ng/ml)-induced HAVIC calcification. These results suggest that MK-4, namely vitamin K2, accelerates calcification of HAVICs from patients with AVS like WFN via PXR-BMP2-ALP pathway. SIGNIFICANCE STATEMENT: For aortic valve stenosis (AVS) induced by irreversible valve calcification, the most effective treatment is surgical aortic or transcatheter aortic valve replacement, but ∼20% of patients are deemed unsuitable because of its invasiveness. For effective drug treatment strategies for AVS, the mechanisms underlying aortic valve calcification must be elucidated. Here, we show that menaquinone-4 accelerates warfarin-induced calcification of AVS-patient human aortic valve interstitial cells in high inorganic phosphate medium; this effect is mediated by pregnane X receptor-bone morphogenetic protein 2-alkaline phosphatase signaling, which could be targeted for novel drug development.

Correction to: Warfarin calcifies human aortic valve interstitial cells at high-phosphate conditions via pregnane X receptor.

In the original publication of the article, part of Fig. 1 was published incorrectly.

Warfarin calcifies human aortic valve interstitial cells at high-phosphate conditions via pregnane X receptor.

Warfarin, a vitamin K antagonist, is the most common anticoagulant used to prevent thromboembolisms associated with atrial fibrillation or following valvular surgery. Although several studies have revealed that long-term warfarin use accelerates aortic valve calcification and the development of aortic stenosis (AS), the detailed mechanism for this phenomenon remains unclear. Therefore, our aim was twofold: to establish the conditions for warfarin-induced calcification of human aortic valve interstitial cells (HAVICs) using high-inorganic phosphate (Pi) conditions and to investigate the underlying mechanism. We prepared and cultured HAVICs from aortic valves affected by calcific aortic valve stenosis (AS group) and aortic valves affected by aortic regurgitation but without any signs of calcification (non-AS group). Under Pi concentrations of 3.2 mM, warfarin significantly increased the calcification and alkaline phosphatase (ALP) activity of AS but not non-AS group HAVICs. Furthermore, gene expression of bone morphogenetic protein 2 (BMP2), a calcigenic marker, was significantly increased following 7 days of warfarin treatment. Warfarin-induced calcification of AS group HAVICs at 3.2 mM Pi was significantly inhibited by dorsomorphin, a Smad inhibitor, and the pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol, but was unaffected by SN-50, an NF-κB inhibitor. Warfarin was also able to increase BMP2 gene expression at a physiological Pi concentration (1.0 mM). Furthermore, excess BMP2 (30 ng/mL) facilitated warfarin-induced ALP upregulation and HAVIC calcification, an effect which was significantly reduced in the presence of coumestrol. Together, our results suggest that warfarin accelerates calcification of HAVICs from AS patients via the PXR-BMP2-ALP pathway.

Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves.

Calcified aortic valve stenosis (CAS) is a common heart valve disease in elderly people, and is mostly accompanied by ectopic valve calcification. We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from CAS patients. In this study, we investigated the role of matrix Gla protein (MGP), a known calcification inhibitor that antagonizes bone morphogenetic protein 2 (BMP2) in TNF-α-induced calcification of HAVICs. HAVICs isolated from aortic valves were cultured, and calcification was significantly induced with 30 ng/mL TNF-α. Gene expression of the calcigenic marker, BMP2, was significantly increased in response to TNF-α, while the gene and protein expression of MGP was strongly decreased. To confirm the role of MGP, MGP-knockdown HAVICs and HAVICs overexpressing MGP were generated. In HAVICs, in which MGP expression was inhibited by small interfering RNA, calcification and BMP2 gene expression were induced following long-term culture for 32 days in the absence of TNF-α. In contrast, HAVICs overexpressing MGP had significantly decreased TNF-α-induced calcification. These results suggest that MGP acts as a negative regulator of HAVIC calcification, and as such, may be helpful in the development of new therapies for ectopic calcification of the aortic valve.

1-Methyl-2-undecyl-4(1H)-quinolone, a derivative of quinolone alkaloid evocarpine, attenuates high phosphate-induced calcification of human aortic valve interstitial cells by inhibiting phosphate cotransporter PiT-1.

An abnormally high serum phosphate level induces calcific aortic stenosis (CAS), which is characterized by ectopic valve calcification and stenosis of the orifice area. Inhibition of ectopic calcification is a critical function of any internal medical therapy for CAS disease. The aim of the present study was to investigate the inhibitory effects of several derivatives of evocarpine, methanolic extracts from the fruits of Evodia rutaecarpa Bentham (Japanese name: Go-Shu-Yu) on the high phosphate-induced calcification of human aortic valve interstitial cells (HAVICs) obtained from patients with CAS. High phosphate (3.2 mM) concentrations significantly increased the calcification of HAVICs after 7 days of culture. This calcification was completely inhibited in the presence of sodium phosphonoformate (PFA), a selective inhibitor of the type III sodium-dependent phosphate cotransporter (PiT-1). PiT-1 contributes to phosphate uptake, resulting in calcification. 1-Methyl-2-undecyl-4(1H)-quinolone (MUQ; 30-300 nM), but not evocarpine or its derivatives dihydroevocarpine and 1-methyl-2-nonyl-4(1H)-quinolone, inhibited the high phosphate-induced HAVICs calcification in a concentration-dependent manner. Although all of the evocarpine derivatives attenuated alkaline phosphatase activity, only MUQ also decreased PiT-1 gene expression with cellular PiT-1 protein diminution. These results suggest that MUQ mitigated high phosphate-induced HAVICs calcification by inhibiting PiT-1 gene expression.

Direct ablation and excision of myocardial scar in post-myocarditis ventricular aneurysm.

The patient was a 34-year-old woman who developed multiple post-myocarditis ventricular aneurysms with ventricular tachyarrhythmia. After implantation of an intracardiac defibrillator, she experienced multiple episodes of counter-shock. An electrophysiological study demonstrated an early excitation circuit entering the septal aneurysm with the right ventricular aneurysm as an exit. A surgical ablation of the re-entry and left ventricular plasty with scar resection was performed. The operation was performed under direct, epicardial electrophysiological guidance. A cryoablation was performed along the right ventriculotomy and the margin of the left ventricular aneurysm. The left ventricle was closed using endoventricular aneurysmorrhaphy and the right ventriculotomy was closed directly. No recurrence of ventricular tachyarrhythmia has been encountered.

A Case of Periaortic Lymphoma Mimicking Complicated Type B Acute Aortic Dissection: A Pitfall in the Endovascular Surgery Era.

We report a case of periaortic lymphoma mimicking Stanford type B acute aortic dissection treated for impending rupture by thoracic endovascular aortic repair. Although no endoleak was detected, the aneurysm enlarged continuously. Repeat computed tomography scans showed that an aortic aneurysm-like structure around the stent graft had enlarged irregularly. Histopathological examination revealed diffuse large B-cell malignant lymphoma. Post-chemotherapy, the aneurysm-like structure disappeared without any fistula or rupture. In open surgery, differentiating between aneurysms and malignancy is easy under direct vision; however, in the endovascular surgery era, this is a pitfall because no surgical specimen of the lesion can be obtained.

Two Surgical Patients with Paget-Schroetter Syndrome Presenting with Atypical Pathophysiology: Non-Effort Thrombosis.

Paget-Schroetter syndrome (PSS) is an upper extremity thrombosis occurring in the axillary and subclavian veins. PSS is also known as "effort thrombosis," because it is usually associated with repetitive and strenuous activities of the upper limbs. We present 2 patients with atypical PSS, so-called "non-effort thrombosis," who were not involved in vigorous activities. They underwent thoracic outlet decompression through the infraclavicular approach without concomitant venoplasty. They were discharged without postoperative anticoagulant therapy. Venography and computed tomography after surgery revealed successful recanalization of the subclavian vein in each case. We highlight the characteristic pathophysiology of "non-effort thrombosis," an atypical PSS entity.

Endovascular Treatment for a Perforated Superficial Femoral Vein Graft for an Infected Abdominal Aortic Aneurysm.

The patient had an infected abdominal aortic aneurysm. Aneurysmectomy and in situ reconstruction of the abdominal aorta using the bilateral superficial femoral veins with omentopexy were performed. On postoperative day 18, hematemesis and melena occurred. Computed tomography showed extravasation from the right graft limb, and gastrointestinal endoscopy showed a duodenal fistula. Covered stent implantation was performed for the limb. Bleeding from the contralateral graft limb occurred the next day, and the patient underwent covered stent graft implantation for that limb followed by partial duodenectomy and duodenojejunostomy. Intraoperative findings demonstrated a pinhole leakage from the branch ligation site on both vein grafts.

Selection and Determination of Treatment for the Spontaneous Isolated Dissection of the Superior Mesenteric Artery.

Objective: This study aimed to clarify the selection and determination of appropriate treatment for acute symptomatic spontaneous isolated dissection of the superior mesenteric artery (SIDSMA). Methods: Data from 10 consecutive patients, who were diagnosed with symptomatic SIDSMA using computed tomography angiography and were managed in our hospital from January 2010 to October 2015, were retrospectively collected and analyzed. Results: There were nine males and one female; mean patient age was 50.3 (range, 35-64) years. All patients experienced acute abdominal pain, and three patients experienced concomitant vomiting. Only one patient exhibited symptoms of suspected peritonitis and intestinal ischemia. Three patients showed improved abdominal pain before admission to our hospital. One patient experienced severe abdominal pain that could not be managed using morphine; he underwent right external iliac to superior mesenteric artery bypass with a great saphenous vein graft. No patient presented with intestinal necrosis. All patients survived, and no patient developed complications during the follow-up period of up to 42 (24.5±16.5) months. Conclusion: Conservative management appears to be the most feasible treatment for SIDSMA. However, open surgery can be performed in patients presenting with any symptoms of intestinal ischemia.

Management of Pulmonary Thromboembolism Based on Severity and Vulnerability to Thrombolysis.

Background: The incidence of pulmonary thromboembolism has been considered rare in Japan. However, its occurrence has been increasing because of westernized lifestyle and diet, increased diagnostic technique, and recognition of this disease. Method: Between January 2003 and September 2014, 179 patients were treated for pulmonary thromboembolism. We classified these patients into 3 groups; Massive (n=35), Sub-massive (n=29) and Nonmassive (n=115) and retrospectively reviewed the treatment options and the outcome. Results: Percutaneous cardiopulmonary support (PCPS) was applied for patients with hemodynamic instability and IVC filter was inserted if there was proximal DVT. In non-massive group (n=115), 95.7% of the patient underwent anticoagulant therapy and 47.0% of the patients received IVC filter. In submassive group (n=29), 48.3% of the patient received thrombolytic therapy and 93.1% of the patient underwent IVC filter insertion. Surgical pulmonary embolectomy was performed in 3 patients who had high risk of thrombolytic therapy in submassive group. There was no death in this group. In massive group, 4 patients who had cardiogenic shock died in acute phase. PCPS was applied in 5 patients, pulmonary embolectomy was performed in 13 patients, thrombolytic therapy was performed in 4 patients and 13 patients underwent anticoagulant therapy alone. There were 7 deaths (20.0%) in this group. Conclusions: In submassive group, treatment should be decided depending on the bleeding risk. In massive group, pulmonary embolectomy was effective. (This is a translation of Jpn J Phlebol 2016; 27: 53-59.).

Management of deep vein thrombosis and pulmonary embolism (venous thromboembolism) during pregnancy.

PURPOSE: The risk of venous thromboembolism (VTE) is high during pregnancy. Although most patients with VTE are safely treated via medications, the optimal treatment for massive pulmonary embolism remains controversial. To evaluate the safety and efficacy of VTE management during pregnancy, we report our single center experience of treating VTE in pregnant women. METHODS: Case records were retrospectively reviewed from seven patients who underwent treatment for venous thromboembolism between 2002 and 2014. RESULTS: Mean gestational time was 28 ± 6.2 weeks. Four patients with deep vein thrombosis were treated medically, and they all had vaginal delivery at full term without hemorrhagic complication. Three patients with massive pulmonary embolism underwent surgical embolectomy. Two of these three patients underwent cesarean delivery at 28 and 29 weeks respectively. There was no maternal death, but one fetal death occurred during surgical embolectomy. CONCLUSION: VTE during pregnant women is safely managed by anticoagulant therapy. Massive pulmonary embolism during pregnancy can be managed safely by surgical embolectomy using cardiopulmonary bypass, but the rate of fetal loss remains high.

Acute limb ischemia: contemporary approach.

Acute limb ischemia is a critical condition with high mortality and morbidity even after surgical or endovascular intervention. Early recognition is important, but a delayed presentation is not uncommon. Viability of the limb is assessed by motor and sensory function and with interrogating Doppler flow signals in pedal arteries and popliteal veins as categorized by Rutherford. Category IIa indicates mild-to-moderate threat to limb salvage over a time frame without revascularization. Limb ischemia is critical without prompt revascularization in category IIb. Because the risk of reperfusion injury is high in this group of patients, perioperative management is important. In category III, reperfusion is not indicated except for embolism within several hours of onset. Intimal injury should be avoided by careful tactile control of a balloon with a smaller size catheter and under radiographic monitoring. Adjunctive treatment with catheter-directed thrombolysis or bypass surgery is sometimes necessary. Endovascular treatment is a promising option for thrombotic occlusion of an atherosclerotic artery. Ischemia-reperfusion injury is a serious problem. Controlled reperfusion with low-pressure perfusion at a reduced temperature and use of a leukocyte filter should be considered. The initial reperfusate is hyperosmolar, hypocalcemic, slightly alkaline, and contains free radical scavengers such as allopurinol. Immediate hemodialysis is necessary for acute renal injury caused by myoglobinemia. Compartment syndrome should be managed with assessment of intra-compartment pressure and fasciotomy.

Reoperative coronary artery bypass surgery: avoiding repeat median sternotomy.

BACKGROUND: Mortality and morbidity in reoperative coronary artery surgery are considered to be higher than those for initial surgery. Contributing factors include cardiac injury and damage to patent grafts in repeat median sternotomy. To avoid these complications, reoperative cases were performed off pump to avoid repeat median sternotomy. METHODS: The study subjects were 79 patients who underwent reoperations while utilizing off-pump coronary artery bypass grafting to avoid the need for repeat median sternotomies. All operations were performed by the same surgeon in the period from January 1996 to December 2010. RESULTS: The mean duration from initial surgery to reoperation was 6 years and 5 months. Reported reasons for reoperation were de novo coronary lesion in 16 patients, graft failure in 33 patients, and de novo coronary lesion plus graft failure in 47 patients. All cases underwent surgery off pump. The approach was left anterior small thoracotomy (35 patients), transdiaphragmatic approach (21 patients), left posterolateral thoracotomy (9 patients), left anterior small thoracotomy plus transdiaphragmatic approach (9 patients), left posterolateral thoracotomy plus transdiaphragmatic approach (4 patients), and small median sternotomy plus left anterior small thoracotomy (1 patient). There were no deaths among the 79 patients in whom repeat median sternotomy was avoided, and all grafts were patent. CONCLUSIONS: Reoperative coronary artery surgery that avoids repeat median sternotomy can prevent cardiac injury and damage to patent grafts. Furthermore, it does not require blood transfusion. Thus, it is an effective method of reducing mortality and morbidity even in reoperative cases.