Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland.

Objective: To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Materials and Methods: Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Results: Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Conclusions: Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.

Switching of cargo sorting from the constitutive to regulated secretory pathway by the addition of cystatin D sequence in salivary acinar cells.

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.

Impending rupture of saphenous vein graft aneurysm with floating fractured bare metal stent treated by coil embolization and covered stent implantation.

Aneurysmal degeneration of a saphenous vein graft (SVG) is a rare, but potentially fatal complication of coronary artery bypass graft (CABG) surgery. In this case report, a patient that had undergone prior CABG surgery and bare metal stent (BMS) implantation at the site of a stenotic SVG lesion presented at our hospital with chest pain, and an SVG aneurysm was detected at the previous BMS implantation site. In addition, the implanted BMS was fractured and floating in the SVG aneurysm. The SVG aneurysm was successfully occluded by percutaneous intervention, using a combination of distal covered stent deployment at the site of the anastomosis between the native coronary artery and the SVG and proximal coil embolization of the aneurysm.

Virtual pathology of cervical radiculopathy based on 3D MR/CT fusion images: impingement, flattening or twisted condition of the compressed nerve root in three cases.

BACKGROUND: There have been several imaging studies of cervical radiculopathy, but no three-dimensional (3D) images have shown the path, position, and pathological changes of the cervical nerve roots and spinal root ganglion relative to the cervical bony structure. The objective of this study was to introduce a technique that enables the virtual pathology of the nerve root to be assessed using 3D magnetic resonance (MR)/computed tomography (CT) fusion images that show the compression of the proximal portion of the cervical nerve root by both the herniated disc and the preforaminal or foraminal bony spur in patients with cervical radiculopathy. FINDINGS: MR and CT images were obtained from three patients with cervical radiculopathy. 3D MR images were placed onto 3D CT images using a computer workstation. The entire nerve root could be visualized in 3D with or without the vertebrae. The most important characteristic evident on the images was flattening of the nerve root by a bony spur. The affected root was constricted at a pre-ganglion site. In cases of severe deformity, the flattened portion of the root seemed to change the angle of its path, resulting in twisted condition. CONCLUSIONS: The 3D MR/CT fusion imaging technique enhances visualization of pathoanatomy in cervical hidden area that is composed of the root and intervertebral foramen. This technique provides two distinct advantages for diagnosis of cervical radiculopathy. First, the isolation of individual vertebra clarifies the deformities of the whole root groove, including both the uncinate process and superior articular process in the cervical spine. Second, the tortuous or twisted condition of a compressed root can be visualized. The surgeon can identify the narrowest face of the root if they view the MR/CT fusion image from the posterolateral-inferior direction. Surgeons use MR/CT fusion images as a pre-operative map and for intraoperative navigation. The MR/CT fusion images can also be used as educational materials for all hospital staff and for patients and patients' families who provide informed consent for treatments.

Long-term adverse effects of cyclophosphamide on follicular growth and angiogenesis in mouse ovaries.

The adverse effects of the anti-cancer agent cyclophosphamide (CTX) on follicular growth and ovarian angiogenesis were investigated in mice. CTX treatment irreversibly induced a loss of follicles through apoptosis and decreased microvascularization of the corpora lutea and follicles in a dose-dependent manner. Our findings demonstrated that CTX adversely affected the ovaries indicating the need to support an awareness of fertility preservation before chemotherapy is initiated.

Oophorectomy for fertility preservation via reduced-port laparoscopic surgery.

BACKGROUND: For fertility preservation of women patients scheduled to undergo chemotherapy or radiotherapy, unilateral oophorectomy was performed, and the ovary was cryopreserved. METHODS: Two-port surgery was conducted in 3 patients, and single-port surgery using a single-incision laparoscopic surgery port in 3. An 18-G Cathelin needle equipped with a syringe was directly inserted transabdominally to reach the small follicle on the ovarian surface; then, follicular fluid was recovered by aspiration through the syringe as with in vitro fertilization procedures, and immature oocytes were collected from the resulting culture medium under microscopy and cryopreserved. Vitrification of the ovarian tissue was performed using the cryotissue method. RESULTS: The operative time and estimated blood loss were 39.7 minutes (17-57) and 8.6 mL (2-20), and the numbers of ovarian cortical tissues and immature oocytes collected were 10.1 (5.5-15) and 16.3 (0-36), respectively. CONCLUSIONS: It is suggested that fertility preservation operations before chemotherapy or radiotherapy can be safely done using reduced-port surgery.

The restorative effects of adipose-derived mesenchymal stem cells on damaged ovarian function.

The clinical application of human adipose-derived mesenchymal stem cells (MSCs) as treatment for intractable diseases or traumatic tissue damage has attracted attention. To address the ability of reactivating injured ovaries, we prepared a rat model with damaged ovaries by using an anticancer agent, cyclophosphamide (CTX). We then investigated the restorative effects on ovarian function and the safety of adipose-derived MSCs (A-MSCs). MSCs were shown to be capable of inducing angiogenesis and restoring the number of ovarian follicles and corpus lutea in ovaries. No deformities, tumor formation or deaths were observed in F1 and F2 rats, indicating that the local injection of MSCs into the ovary did not have any obvious side effects. In addition, the localization of the Y chromosome was investigated using the fluorescent in situ hybridization method by injecting male A-MSCs into the ovaries; as a result, the Y chromosomes were localized not in the follicles, but in the thecal layers. ELISA revealed that A-MSCs secreted higher levels of vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) than tail fibroblast cells. Quantitative real-time PCR and immunohistochemistry showed that higher expression levels of VEGF, IGF-1 and HGF were observed in CTX-treated ovaries after A-MSC transplantation. These findings suggest that MSCs may have a role in restoring damaged ovarian function and could be useful for regenerative medicine.

Uterine autotransplantation in cynomolgus macaques: the first case of pregnancy and delivery.

BACKGROUND: For women with congenital uterine infertility, or for those who have undergone hysterectomy, uterine transplantation is one of the potential treatments to regain fertility. In this study, we utilized a primate model of uterine transplantation, and evaluated the patency of our microsurgical anastomoses, and the perfusion of the transplanted uterus. METHODS: Two female cynomolgus monkeys underwent surgery. We anastomosed two arteries and one vein in Case 1 and two arteries and two veins in Case 2. The arteries used were the uterine arteries and the anastomosis was done to the external iliac artery. We used one of the ovarian veins in both animals, but resected the ovary from the Fallopian tube. Uterine arterial blood flow and uterine size were determined by intraoperative indocyanine green (ICG) angiography and ultrasonography. The biopsy of the uterine cervix was performed after surgery. RESULTS: ICG angiography showed that the unilateral uterine artery perfused the bilateral uterine bodies and cervix. In Case 1, ICG angiography showed the occlusion of one of the anastomosed arteries during the operation and the uterus appeared atrophied 2 months after operation. In Case 2, the transplanted uterus survived and normal menstruation occurred. The animal achieved a natural pregnancy and was delivered by the Caeserean section due to early separation of the placenta. The newborn suffered fetal distress. CONCLUSIONS: These results show the anastomosis of at least the bilateral uterine arteries and the unilateral ovarian vein is required for uterus transplantation. This is the first report of a natural pregnancy in a primate following uterine autotransplantation.

Surgical procedure to conserve the uterus for future pregnancy in patients suffering from massive adenomyosis.

The treatment for severe adenomyosis has usually been hysterectomy, because there is no line of demarcation between diseased and normal tissue. Yet many such women wish to retain their uterus and some even wish to bear children. This report evaluates the efficacy of a new method of adenomyomectomy, where adenomyotic tissues are radically excised and the uterine wall is reconstructed by a triple-flap method, without overlapping suture lines, to prevent uterine rupture in subsequent pregnancies. This is a prospective case series followed for 10 years from June 1998 to August 2008 of 104 women with severe adenomyosis verified histologically and with magnetic resonance imaging. There was a dramatic reduction in both dysmenorrhoea and hypermenorrhoea and all patients returned to having normal menstrual cycles. Of 26 women who wished to conceive, 16 became pregnant, 14 (53.8%)went to term and delivered a healthy baby and there were no cases of uterine rupture. Adenomyosis symptoms recurred in only four out of 104 cases. The procedure thus resulted in a dramatic reduction in symptoms and allowed over half of women who wished to conceive to go to term without uterine rupture.

Thiol-oxidation reduces the release of amylase induced by ß-adrenergic receptor activation in rat parotid acinar cells.

In parotid acinar cells, the activation of ß-adrenergic receptors induces the accumulation of intracellular cAMP, and consequently provokes the exocytotic release of amylase, a digestive enzyme. The cellular redox status plays a pivotal role in regulating various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damage. In this study, we examined the effects of diamide, a thiol-oxidizing reagent, on amylase release by rat parotid acinar cells. In cells treated with diamide, the formation of cAMP and the release of amylase induced by the ß-agonist isoproterenol (IPR) were partially reduced. The inhibitory effect of diamide on the IPR-induced release of amylase could be abrogated by reduced glutathione or dithiothreitol. Diamide had no effect on the amylase release induced by forskolin, an adenylate cyclase activator, or by mastoparan, a heterotrimeric GTPbinding protein activator. In cells treated with diamide, the binding affinity for [(3)H]DHA, but not the number of binding sites, was reduced. These results suggest that ß-adrenergic receptor function is reduced by thiol-oxidation, which inhibits amylase secretion by parotid acinar cells.

[A case of hard metal lung disease in a man who worked as an iron grinder].

The patient was a man who had suffered from repeated pneumothoraces since August 2003, when he was 16 years old. A right pneumothorax was observed at age 21 years, in April 2008. At the same time, a dry cough began to appear and diffuse small nodular shadows in both lung fields were found on a chest X-ray film. Due to worsening symptoms and the chest X-ray findings, a transbronchial lung biopsy was performed in September 2008. Pathological examination showed mural type organization, and large numbers of multinucleated giant cells that were engulfing nucleated cells and had black pigment in their cytoplasm. Giant cell interstitial pneumonia and hard metal lung disease (HMLD) were suspected because of the patient's occupational history as a metal grinder, which included the use of a hard metal tool for three years since August 2005. In an elementary analysis using an electron probe microanalyzer, tungsten was detected in resected lung tissue obtained in April 2008 which confirmed the diagnosis. His symptoms improved after the initiation of corticosteroid therapy, which continued but with a gradual decrease in the dose. In this case, HMLD developed over a relatively short period despite the low level of dust dispersal of a hard-metal tool, perhaps because of a hypersensitivity of the patient to hard metal.

The thiol-oxidizing agent diamide reduces isoproterenol-stimulated amylase release in rat parotid acinar cells.

In parotid acinar cells, activation of beta-adrenergic receptors provokes exocytotic amylase release via the accumulation of intracellular cAMP. Cellular redox status plays a pivotal role in the regulation of various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damages. In this study, we examined effect of diamide, a thiol-oxidizing reagent, on amylase release in rat parotid acinar cells. In the presence of diamide, isoproterenol (IPR)-induced cAMP formation and amylase release were partially reduced. Diamide had no effect on amylase release induced by forskolin and mastoparan, an adenylate cyclase activator and heterotrimeric GTP binding protein activator, respectively. In the cells pretreated with diamide, the binding affinity of [(3)H]dihydroalprenolol to beta-receptors was reduced. These results suggest that oxidative stress results in reduction of binding affinity of ligand on beta-receptor and consequently reduces protein secretory function in rat parotid acinar cells.

Minimal ovarian stimulation with clomiphene citrate: a large-scale retrospective study.

Enclomiphene, an isomeric component of clomiphene citrate, acts antagonistically to the oestradiol receptor at the hypothalamus level, inhibiting both negative and positive feedback, and resulting in the induction of ovarian stimulation and suppression of ovulation. The minimal ovarian stimulation protocol takes full advantage of these characteristics of clomiphene citrate. Administration of 50 mg clomiphene citrate is initiated on cycle day 3, and from day 8 patients receive 150 IU of FSH every other day. When the size of the dominant follicle and the oestradiol concentration reach the predefined values, gonadotrophin-releasing hormone agonist is administered to induce follicular maturation. Oocytes are then retrieved 32-35 h later. Because the short half-life of enclomiphene (24 h) is of critical importance in this protocol, it is necessary to continue oral administration of clomiphene citrate until the day before maturation is triggered. Of all 43,433 cycles initiated, the rates for oocyte retrieval and embryo cleavage were 83 and 64% respectively. The mean number of oocytes retrieved was 2.2. The rates for live births, miscarriages, and ectopic pregnancies, in relation to initiated cycles, including cases of frozen-thawed transfer, were 11.1, 3.4 and 0.2% respectively.

Bone marrow derived cells are involved in the pathogenesis of cardiac hypertrophy in response to pressure overload.

BACKGROUND: Bone marrow (BM) cells possess broad differentiation potential and can form various cell lineages in response to pathophysiological cues. The present study investigated whether BM-derived cells contribute to the pathogenesis of cardiac hypertrophy, as well as the possible cellular mechanisms involved in such a role. METHODS AND RESULTS: Lethally irradiated wild-type mice were transplanted with BM cells from enhanced green fluorescent protein-transgenic mice. The chimeric mice were subjected to either prolonged hypoxia or transverse aortic constriction. BM-derived enhanced green fluorescent protein-expressing cardiomyocytes increased in number over time, emerging predominantly in the pressure-overloaded ventricular myocardium, although they constituted <0.01% of recipient cardiomyocytes. To determine whether BM-derived cardiomyocytes were derived from cell fusion or transdifferentiation at the single-cell level, lethally irradiated Cre mice were transplanted with BM cells from the double-conditional Cre reporter mouse line Z/EG. BM-derived cardiomyocytes were shown to arise from both cell fusion and transdifferentiation. Interestingly, BM-derived myofibroblasts expressing both vimentin and alpha-smooth muscle actin were concentrated in the perivascular fibrotic area. These cells initially expressed MAC-1/CD14 but lost expression of these markers during the chronic phase, which suggests that they were derived from monocytes. A similar phenomenon occurred in cultured human monocytes, most of which ultimately expressed vimentin and alpha-smooth muscle actin. CONCLUSIONS: We found that BM-derived cells were involved in the pathogenesis of cardiac hypertrophy via the dual mechanisms of cell fusion and transdifferentiation. Moreover, the present results suggest that BM-derived monocytes accumulating in the perivascular space might play an important role in the formation of perivascular fibrosis via direct differentiation into myofibroblasts.

Production of the first offspring from oocytes derived from fresh and cryopreserved pre-antral follicles of adult mice.

Although mammalian ovaries contain hundreds of thousands of pre-antral follicles, fewer than 1% of these reach maturity and ovulation. Obtaining immature eggs from the pre-antral follicles of ovarian tissue could increase the possibility of preserving fertility in women undergoing anti-cancer treatment, and in women who wish to delay pregnancy and child raising until they are older. This study reports the birth of 10 healthy mouse pups derived from oocytes obtained from pre-antral follicles after adult ovary tissue cryopreservation and allotransplantation. High in-vitro maturation (55.1%), fertilization (76.3%) and cleavage (98.3%) rates were achieved using these oocytes, and there was no significant difference between the vitrified and control samples except in maturation rate (55.1 versus 72.8%, P < 0.05). After an ultra-rapid vitrification procedure, the warmed tissue fragments were transplanted beneath the kidney capsule of severe combined immunodeficient mice for onward in-vivo culture. Within 10 days of culture, 138 full size oocytes developed from the 456 transplanted pre-antral follicles. In-vivo growth of follicles was followed by in-vitro oocyte maturation, in-vitro fertilization and subsequent embryo transfer, leading to the birth of 10 healthy pups. These results may lead to increasing the possibility of preserving fertility by cryopreservation of ovarian tissue.

Clinical features of hepatitis C virus-related hepatocellular carcinoma and their association with alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II.

PURPOSE: We investigated the differences in clinical features between alpha-fetoprotein (AFP)-predominant hepatocellular carcinoma (HCC) and protein induced by vitamin K absence or antagonist-II (PIVKA-II)-predominant HCC, especially regarding host factors thought to contribute to hepatocarcinogenesis in chronic hepatitis C virus (HCV) infection. METHODS: HCV-related HCC patients (n=306) were divided into four groups according to median AFP (48.1 ng/ml) and PIVKA-II (60 mAU/ml). Host factors, tumor factors, survival, and risk factors affecting survival were compared. RESULTS: Aspartate aminotransferase (AST; IU/L), alanine aminotransferase (ALT; IU/L), and platelet count (x 10(4)/ml) were, respectively, 81, 67, and 8.2 in AFP-predominant HCC (group A; n=66) vs. 50, 42, and 11.4 in PIVKA-II-predominant HCC (group P; n=52). Tumor sizes (mm) in groups A and P were 20 and 37, respectively. Significant differences were evident. Survival was identical between the two groups. Factors affecting survival were total bilirubin, portal tumor thrombus and number of nodule in group A, and albumin and tumor distribution in group P. CONCLUSIONS: PIVKA-II-predominant HCC had a milder hepatitis and a better-preserved platelet count compared with AFP-predominant HCC. Considering the strong relation between hepatocarcinogenesis and hepatic inflammation with chronic HCV infection, these differences indicate that hepatocarcinogenic mechanisms in PIVKA-II-predominant HCC may differ from those in AFP-predominant HCC.

The efficacy and safety of managing ectopic pregnancies with transvaginal ultrasound-guided local injections of absolute ethanol.

PURPOSE: To describe the efficacy and safety of managing ectopic pregnancies (EP) with ultrasound-guided local injections of absolute ethanol (AE). METHODS: 69 cases of EP following IVF performed in our clinic were treated with a local injection of 0.3 ml AE with a 23-gauge needle under transvaginal ultrasonic guidance. The efficacy was evaluated comparing serum beta-human chorionic gonadotropin (beta-hCG) levels before and after the injection. RESULTS: In the 60 successful cases (87%), the serum beta-hCG level decreased by 10-30% in two hours postinjection. Of these, 46 were effective with a single injection and the half-life of beta-hCG was achieved within 4 days in 45 cases. In 56 cases (including repetitive administration) serum beta-hCG levels decreased to 20 mIU/mL within 20 days. The treatment showed no side effects and could be given on an outpatient basis without anesthesia. CONCLUSIONS: This method was shown to be a safe, effective new approach to treating EP.

Highly efficient vitrification method for cryopreservation of human oocytes.

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

[Case of primary pulmonary cryptococcosis accompanied by severe back pain].

We report a case of primary pulmonary cryptococcosis. A 43-year-old male, without any significant underlying disease or immunological abnormalities, was admitted to our hospital with a complaint of sudden onset of severe back pain. His chest-X-ray and computed tomography revealed infiltrative shadows in the left lower lung without any signs of pleural effusion. Through transbronchial biopsy, cryptococcosis was obtained. Cryptococcal antigen also tested positive, we diagnosed this case as primary pulmonary cryptococcosis. And started anti-fungal therapy (fluconazole) consisting of parenteral and oral fluconazole. As soon as anti-fungal therapy was started, both the chest X-ray findings and cryptococcal antigen showed general improvement. Furthermore, subjective symptoms subsided immediately after treatment, During follow up through the outpatient clinic, his symptomatic complaint and chest roentgenogram shows improvements. This case was noteworthy for two reasons: 1) In cases with chest X-rays showing infiltrative shadows but lacking any underlying diseases, pulmonary cryptococcosis should be considered. 2) Sudden onset of back pain is a rare but a possible primary symptom of pulmonary cryptococcosis.

Partial spontaneous regression of hepatocellular carcinoma: a case with high concentrations of serum lens culinaris agglutinin-reactive alpha fetoprotein.

Spontaneous regression of hepatocellular carcinoma (HCC) is rare. There are few reports discussing spontaneous regression associated with serum lens culinaris agglutinin-reactive alpha fetoprotein (AFP-L3). We describe a case of HCC with a high level of AFP-L3, which showed a rapid increase in alpha fetoprotein (AFP) concentration after partial spontaneous regression, and which was then treated successfully. A 71-year-old woman suffering from chronic hepatitis C underwent surgical resection for HCC. Preoperative concentrations of AFP fluctuated; subsequent to a transient decrease, a rapid increase in AFP was observed. AFP-L3 concentration was extremely high. The resected tissue consisted of encapsulated moderately differentiated HCC, subcapsular coagulation necrosis, and chronic active hepatitis. The postoperative course was uneventful. At present, 24 months after diagnosis, no symptoms or signs of tumor recurrence or metastasis have been observed. Although the precise etiology of the spontaneous regression in this patient is not known, we speculate that spontaneous regression of tumor cells with high malignant potential may be related to arterial involvement and insufficient blood supply.