Relationship between myocardial metabolites and contractile abnormalities during graded regional ischemia. Phosphorus-31 nuclear magnetic resonance studies of porcine myocardium in vivo.

The mechanisms responsible for changes in myocardial contractility during regional ischemia are unknown. Since changes in high-energy phosphates during ischemia are sensitive to reductions in myocardial blood flow, it was hypothesized that myocardial function under steady-state conditions of graded regional ischemia is closely related to changes in myocardial high-energy phosphates. Therefore, phosphorus-31 nuclear magnetic resonance spectroscopy was employed in an in vivo porcine model of graded coronary stenosis. Simultaneous measurements of regional subendocardial blood flow, high-energy phosphates, pH, and myocardial segment shortening were made during various degrees of regional ischemia in which subendocardial blood flow was reduced by 16-94%. During mild reductions in myocardial blood flow (subendocardial blood flow = 83% of nonischemic myocardium), only the ratio of phosphocreatine to inorganic phosphate (PCr/Pi), Pi, and [H+] were significantly changed from control. PCr, ATP, and PCr/ATP were not significantly reduced from control with mild reductions in blood flow. Changes in myocardial segment shortening were most closely associated with changes in PCr/Pi (r = 0.94). Pi and [H+] were negatively correlated with segment shortening (r = -0.64 and -0.58, respectively) and increased over twofold when blood flow was reduced by 62%. Thus, these data demonstrate that PCr/Pi is sensitive to reductions in myocardial blood flow and closely correlates with changes in myocardial function. These data are also consistent with a role for Pi or H+ as inhibitors of myocardial contractility during ischemia.

Energetics of acute pressure overload of the porcine right ventricle. In vivo 31P nuclear magnetic resonance.

In vivo 31P nuclear magnetic resonance (NMR) spectroscopy of the right ventricular (RV) free wall was employed to determine (a) whether phosphorus energy metabolites vary reciprocally with workload in the RV and (b) the mechanisms that limit RV contractile function in acute pressure overload. In 20 open-chest pigs, phosphocreatine (PCr)/ATP ratio (an index of energy metabolism inversely related to free ADP concentration), myocardial blood flow (microspheres), and segment shortening (sonomicrometry, n = 14) were measured at control (RV systolic pressure 31 +/- 1 mm Hg), and with pulmonary artery constriction to produce moderate pressure overload (RV systolic pressure 45 +/- 1 mm Hg), and maximal pressure overload before overt RV failure and systemic hypotension (RV systolic pressure 60 +/- 1 mm Hg). With moderate pressure overload, PCr/ATP declined to 89% of control (P = 0.01), while contractile function increased. Adenosine (n = 10, mean dose 0.16 mg/kg-min) increased RV blood flow by an additional 41% without increasing PCr/ATP, indicating that coronary reserve was not depleted and that the decrease in PCr/ATP from control was not due to ischemia. With maximal pressure overload and incipient RV failure, PCr/ATP fell further to 81% of control and RV blood flow did not increase further, even with adenosine. Thus: (a) The decline in PCr/ATP with moderate RV pressure overload, without evident ischemia or contractile dysfunction, supports the positive regulation of oxidative phosphorylation by ATP hydrolysis products. (b) Depletion of RV coronary flow reserve accompanies the onset of RV failure at maximal pressure overload.

Troglitazone improves recovery of left ventricular function after regional ischemia in pigs.

BACKGROUND: This study determined whether treatment of normal (nondiabetic) pigs with the insulin-sensitizing agent troglitazone improves recovery of left ventricular (LV) function after acute ischemia and whether such effects are associated with altered myocardial substrate metabolism. METHODS AND RESULTS: Juvenile pigs (n=6) were treated with troglitazone (75 mg. kg(-1). d(-1) PO) for 8 weeks. Untreated pigs (n=8) served as controls. Under anesthetized, open-chest conditions, pigs underwent 90 minutes of moderate regional LV ischemia and 90 minutes of reperfusion. Regional LV function was assessed with subendocardial sonomicrometry crystals. Fasting plasma insulin and free fatty acid levels were lower in troglitazone-treated pigs than in untreated pigs, whereas blood glucose did not differ between groups. These findings suggest that treatment enhanced systemic insulin sensitivity. Baseline hemodynamics and regional LV function did not differ between groups. After ischemia and reperfusion, systolic function (external work) of the ischemic region recovered to 44+/-6% of baseline in troglitazone-treated pigs versus 18+/-6% of baseline in untreated pigs (P<0.05). Regional diastolic function (maximum rate of wall expansion) recovered to 78+/-7% of baseline in treated pigs versus 52+/-7% of baseline in untreated pigs (P<0.05). Recovery of global LV systolic and diastolic function was also significantly greater in treated pigs. Myocardial glucose uptake did not differ between groups under any condition; however, net myocardial lactate uptake after reperfusion was 7 times greater in troglitazone-treated pigs than in untreated pigs, suggesting that treatment enhanced myocardial carbohydrate oxidation after reperfusion. CONCLUSIONS: In nondiabetic pigs, chronic troglitazone treatment improves recovery of LV systolic and diastolic function after acute ischemia.

The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer.

Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25-dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3-1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10(-9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer.

Low-dose inotropic stimulation during left ventricular ischaemia does not worsen post-ischaemic dysfunction.

OBJECTIVE: Inotropic agents are used clinically to improve ventricular function during ischaemia. The goal of this study was to determine whether inotropic stimulation during moderate left ventricular (LV) ischaemia exacerbated post-ischaemic LV dysfunction. METHODS: In 18 open-chest, anesthetized pigs, LV pressure versus subendocardial segment length loops were used to generate regional preload-recruitable stroke work (PRSW) and LV end-diastolic pressure (EDP) versus end-diastolic segment length (EDL) relations. Ischaemia was produced by constant, partial constriction of the mid anterior descending coronary artery for 90 min. Nine pigs received dobutamine (4 micrograms.kg-1.min-1, i.v.) during the final 60 min of ischaemia (Group 2), while 9 other pigs did not (Group 1). RESULTS: During unstimulated ischaemia, anterior subendocardial blood flow (Group 1, 0.27 +/- .05; Group 2, 0.30 +/- .07 ml.g-1.min.-1, mean +/- s.e.m.) and steady-state PRSW (Group 1, 30 +/- 4%; Group 2, 27 +/- 5% of baseline) were similar in both groups. Dobutamine stimulation during ischaemia increased heart rate, mean arterial pressure, subendocardial blood flow, oxygen consumption and steady-state PRSW of the ischaemia zone, but not lactate release. After 60 min reperfusion, steady-state ischaemic zone PRSW remained markedly and nearly equally reduced in both groups (Group 1, 28 +/- 4%; Group 2, 23 +/- 5% of baseline). Reduced PRSW after reperfusion was due primarily to persistent rightward shift of the PRSW intercept with only a modest contribution from reduced PRSW slope. CONCLUSIONS: Low-dose inotropic stimulation during moderate regional LV ischaemia increases aerobic, but not anaerobic energy metabolism, and does not worsen post-ischaemic dysfunction.

Right ventricular dysfunction persists following brief right ventricular pressure overload.

OBJECTIVE: Acute pulmonary hypertension may cause right ventricular (RV) contractile failure. While it has been assumed that restoration of normal loading conditions after acute pulmonary hypertension is sufficient for complete recovery of RV function, this has not been rigorously examined. The purpose of this study was to test the hypothesis that acute RV pressure overload produces RV contractile dysfunction that persists following restoration of control loading conditions. METHODS: We subjected 18 autonomically-blocked, chloralose-anesthetized, open-chest pigs to 1 h of pulmonary artery constriction to increase RV systolic pressure from 35 +/- 1 to 55 +/- 1 mmHg, followed by 2 h of measurements after pulmonary artery constriction release. We determined regional RV free wall function from pressure-segment length loops and preload recruitable stroke work relations, and global RV function from stroke work vs. end-diastolic pressure relations. RESULTS: As expected, RV free wall systolic shortening diminished during pulmonary artery constriction, but the endo/epi blood flow ratio, lactate uptake, and coronary venous pH were not significantly changed. Following release of pulmonary artery constriction, RV systolic and diastolic pressure returned to control values. Nonetheless, contractile dysfunction persisted, with depressed RV free wall systolic shortening (70 +/- 22% of control), RV regional external work (59 +/- 11% of control at control end-diastolic length), and global RV stroke work (56 +/- 14% of control at control end-diastolic pressure). Depressed regional work was due to a parallel, rightward shift of the preload recruitable stroke work relation. Five pigs identically instrumented but not subjected to pulmonary artery constriction showed no significant over 3 h. CONCLUSIONS: Acute pulmonary hypertension causes RV contractile dysfunction that persists at least 2 h after restoration of control loading conditions. Contractile dysfunction is not attributable to RV ischemia during pressure overload.

Effect of aminophylline on coronary reactive and functional hyperaemic response in conscious dogs.

This study was undertaken to determine whether adenosine release from myocardial cells plays a role in coronary reactive and functional vasomotion. Reactive hyperaemic blood flow responses to 10 s complete occlusions and 400 ms diastolic occlusions of the left circumflex coronary artery and to the vasomotor responses to the increased cardiac demand following ventricular extra-activation were examined in a chronic, heart-blocked dog preparation during a control period and following intravenous bolus administration of aminophylline (5 mg X kg-1). Aminophylline administration resulted in a 19% decrease in the blood flow debt repayment ratio of 10 s reactive hyperaemic responses compared with the control period. However, administration of aminophylline had no effect on the coronary vascular response to 400 ms diastolic occlusions or to ventricular extra-activations. These observations indicate that adenosine may play a role in the coronary vascular response to prolonged interventions but that other factors, as yet unidentified, may be implicated in the beat-to-beat regulation of coronary vascular resistance.

Effect of supranormal coronary blood flow on energy metabolism and systolic function of porcine left ventricle.

OBJECTIVE: The goal was to determine if supranormal coronary blood flow increases myocardial oxygen consumption, high energy phosphate levels, and systolic function in the in situ autoperfused heart. METHODS: Thirteen anaesthetised open chest pigs with an intact, autoperfused coronary circulation, weight 30-40 kg, were studied. Measurements were made under basal conditions and during regional hyperperfusion of the anterior left ventricle produced by intracoronary infusion of adenosine (mean dose 3.3 mumol.min-1). Doppler coronary blood flow velocity in the anterior descending coronary artery, arterial and anterior interventricular venous blood oxygen content, high energy phosphates (by transmurally localised 31P NMR), and myocardial wall thickening (by sonomicrometry) were measured. RESULTS: With adenosine, coronary flow was increased to 355(SEM 59)% of control. Supranormal coronary flow produced no significant changes in anterior left ventricular oxygen consumption [99(12)% of control]. 31P NMR spectroscopy revealed no significant changes in the peak intensities of phosphocreatine or ATP in either the subendocardium or subepicardium (90-97% of control). Systolic anterior left ventricular wall thickening also did not change [107(13)% of control]. CONCLUSIONS: Supranormal coronary flow does not augment myocardial oxygen consumption, high energy phosphates, or systolic function in the in situ autoperfused heart. Myocardial oxygen delivery does not limit oxidative metabolism under normal conditions.

Multiple myeloma: clusters, clues, and dioxins.

Multiple myeloma (MM) is a B-cell neoplasm of unknown etiology. We searched for etiological clues by examining the literature on geographic clusters of MM. We searched the MEDLINE database from 1966 to 1996 for spatial occurrences of MM that were significantly greater than expected (spatial "clusters"). Eight clusters with verified diagnoses of MM were identified. All of the eight clusters occurred in locations that were proximate to a body of water. Six of these bodies of water are known to have been contaminated with dioxins. We hypothesize that the observed association between MM and proximity to bodies of water is caused by exposure to dioxins in individuals who consume local fish and seafood. This hypothesis is consistent with the significantly elevated risks for MM in groups with high consumption of dioxin-contaminated fish, e.g., Baltic Sea fishermen and Alaskan Indians, and among persons accidentally exposed to dioxins in Seveso, Italy. Dioxins are immunotoxic and inhibit the differentiation of B cells. Thus, dioxins are plausible myelomagens. A dioxin hypothesis could illuminate many epidemiological features of MM and may suggest new avenues for analytic research.

Is cadmium a cause of human pancreatic cancer?

Little is known about the etiology of pancreatic cancer, which is an important cause of cancer mortality in developed countries. We hypothesize that exposure to cadmium is a cause of pancreatic cancer. Cadmium is a nonessential metal that is known to accumulate in the human pancreas. The major risk factors for pancreatic cancer (increasing age, cigarette smoking, residence in Louisiana, and occupations involving exposure to metalworking and pesticides) are all associated with increased exposure to cadmium. Our meta-analysis of cohorts with high exposure to cadmium is also consistent with an increased risk of pancreatic cancer (standardized mortality ratio = 166; 95% confidence interval, 98-280; P = 0.059). Cadmium can cause the transdifferentiation of pancreatic cells, increases in the synthesis of pancreatic DNA, and increases in oncogene activation. Thus, cadmium is a plausible pancreatic carcinogen. The cadmium hypothesis provides a coherent explanation for much of the descriptive epidemiology of pancreatic cancer and suggests new avenues for analytical research.

Vitamin D and breast cancer risk: the NHANES I Epidemiologic follow-up study, 1971-1975 to 1992. National Health and Nutrition Examination Survey.

We analyzed data from the first National Health and Nutrition Examination Survey Epidemiologic Follow-up Study to test the hypothesis that vitamin D from sunlight exposure, diet, and supplements reduces the risk of breast cancer. We identified 190 women with incident breast cancer from a cohort of 5009 white women who completed the dermatological examination and 24-h dietary recall conducted from 1971-1974 and who were followed up to 1992. Using Cox proportional hazards regression, we estimated relative risks (RRs) for breast cancer and 95% confidence intervals, adjusting for age, education, age at menarche, age at menopause, body mass index, alcohol consumption, and physical activity. Several measures of sunlight exposure and dietary vitamin D intake were associated with reduced risk of breast cancer, with RRs ranging from 0.67-0.85. The associations with vitamin D exposures, however, varied by region of residence. The risk reductions were highest for women who lived in United States regions of high solar radiation, with RRs ranging from 0.35-0.75. No reductions in risk were found for women who lived in regions of low solar radiation. Although limited by the relatively small size of the case population, the protective effects of vitamin D observed in this prospective study are consistent for several independent measures of vitamin D. These data support the hypothesis that sunlight and dietary vitamin D reduce the risk of breast cancer.

25-Hydroxyvitamin D3, the prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic epithelial cells.

The hormonal metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] is known to inhibit the proliferation of prostatic epithelial cells. This has stimulated interest in vitamin D compounds as therapeutic agents for prostate cancer. However, the therapeutic use of 1,25(OH)2D3 is limited because elevations in serum 1,25(OH)2D3 can cause dangerous elevations in serum calcium levels. We wondered whether the prohormone of 1,25(OH)2D3, 25-hydroxyvitamin D3 (25-OH-D3), which is much less calcemic, could also achieve antiproliferative effects in prostatic cells. 25-OH-D3 is converted to 1,25(OH)2D3 by the mitochondrial enzyme 1-alpha-hydroxylase. We have recently shown that human prostatic cells also possess significant 1-alpha-hydroxylase activity (Schwartz et al., Cancer Epidemiol. Biomark. Prev., 7: 391-395, 1998). We studied 1-alpha-hydroxylase gene expression in four strains of primary human prostatic epithelial cells by reverse transcription PCR amplification (RT-PCR) of 1-alpha-hydroxylase. Human prostatic stromal cells were negative for 1-alpha-hydroxylase by RT-PCR. This led us to hypothesize that 25-OH-D3 would inhibit the proliferation of prostatic epithelial cells because 25-OH-D3 would be converted to 1,25(OH)2D3 intracellularly. We studied the effects of 25-OH-D3 and 1,25(OH)2D3 on the proliferation of prostatic epithelial cells using high density growth and clonal growth assays on two different primary cell strains derived from normal human prostatic peripheral zone. 25-OH-D3 and 1,25(OH)2D3 each inhibited growth in a dose- and time-dependent manner. Growth inhibition was evident at 1 nM, and maximal inhibition was observed at 100 nM within 10-12 days of exposure. The potencies of 25-OH-D3 and 1,25(OH)2D3 were not significantly different. These data demonstrate that 25-OH-D3, which previously was thought to have little biological activity, can become a potent antiproliferative hormone for prostatic cells that express 1-alpha-hydroxylase. Because 25-OH-D3 exhibits similar potency to 1,25(OH)2D3 but is less calcemic, 25-OH-D3 may offer a safer option than 1,25(OH)2D3 for prostate cancer therapy. Moreover, because 25-OH-D3 is produced endogenously from vitamin D, these findings support a potential role for vitamin D in the chemoprevention of prostate cancer.

Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3.

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.

1 alpha,25-Dihydroxyvitamin D (calcitriol) inhibits the invasiveness of human prostate cancer cells.

1 alpha,25-Dihydroxyvitamin D (1,25 D; also know as calcitriol), the hormonal form of vitamin D, can inhibit the proliferation and promote the differentiation of human prostate adenocarcinoma cells. However, little is known about the effects of 1,25 D on the invasive ability of prostate cancer cells. We used an in vitro bioassay of cell invasion (Amgel assay) to examine the effects of 1,25 D and a "noncalcemic" vitamin D analogue, 1,25-dihydroxy-16-ene-23-yne-cholecalciferol (16-23-D3), on the invasiveness of three well-characterized human prostate carcinoma cell lines: DU 145, PC-3, and LNCaP. PC-3 and LNCaP cells were poorly invasive in Amgel and were hardly affected by treatment with 1,25 D or 16-23-D3 (< 3%). Conversely, DU 145 cells were highly invasive in Amgel, and their invasion was markedly inhibited by 1,25 D and 16-23-D3 (maximally 66 and 59.4% respectively). This effect was both dose-dependent, with maximal inhibition at 1 x 10(-7) M and 72 h. Significant inhibition of invasion was observed at physiological levels of 1,25 D. Neither proliferative indices nor cell cycle kinetics were altered during the experimental exposure. Treatment with 1,25 D and 16-23-D3 caused a selective decrease in the secreted levels of type IV collagenases (MMP-2 and MMP-9). These findings support the hypothesis that 1,25 D reduces the risk of invasive prostate cancer and suggest a role for vitamin D compounds in the chemoprevention of invasive prostate cancer.

Inhibition of prostate cancer metastasis in vivo: a comparison of 1,23-dihydroxyvitamin D (calcitriol) and EB1089.

The steroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] is known to inhibit the proliferation and to promote the differentiation of human prostate cancer cells. Additionally, we showed that 1,25(OH)2D markedly inhibits the invasiveness of human prostate cancer cells in vitro (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997). These properties support the use of 1,25(OH)2D as differentiation therapy in prostate cancer. However, the use of 1,25(OH)2D in vivo is limited by the risk of hypercalcemia. We therefore compared the effects of 1,25(OH)2D and of EB1089, an analogue of 1,25(OH)2D with reduced calcemic effects, in an in vivo model of androgen-insensitive metastatic prostate cancer, the rat Dunning MAT LyLu prostate cancer model. Tumor growth and metastasis were studied using Copenhagen rats given s.c. injections of MAT LyLu cells. Fifty male rats were divided into five groups of 10 rats each. Four experimental groups received i.p. injections of low and high doses of 1,25(OH)2D and EB1089 (0.5 and 1.0 microg/kg, low and high, respectively). A control group received injections of vehicle only. Tumor volumes were measured three times per week. Rats were weighed weekly. The number of metastases to the lungs and the extent of hypercalcemia were evaluated. Compared with controls, tumor volumes were significantly smaller in all experimental groups. Similarly, the number of lung metastases (number of foci/lung) was reduced markedly by both 1,25(OH)2D and EB1089. Control rats developed 22.7 (+/- 1.98 SE) tumor foci per lung. Rats treated with 1,25(OH)2D and with EB1089 (1.0 microg/kg) developed 10.4 (+/- 2.81) and 7.70 (+/- 1.29) tumor foci, respectively (P < 0.001 and P < 0.0001, respectively; drug versus control). Compared with controls (10.79 +/- 0.1 mg/dl), serum calcium levels were significantly elevated in both 1,25(OH)2D and EB1089-treated rats (P < 0.01). However, EB1089 was significantly less calcemic than 1,25(OH)2D (12.59 +/- 0.21 mg/dl versus 14.47 +/- 0.46 mg/dl; 1.0 microg/kg; P < 0.001). Rats treated with 1,25(OH)2D showed marked weight loss: 20.0 +/- 1.9% and 26.3 +/- 1.7% of their initial weight (low and high doses, respectively, P < 0.001). Weight loss was significantly lower in rats treated with EB1089 at the high dose 8.4 (+/- 2.9) %. Moreover, rats treated with low-dose EB1089 gained 5.2 (+/- 3.7) % of their initial weight. In conclusion, 1,25(OH)2D and EB1089 showed marked and equivalent inhibition of prostate cancer metastasis in vivo. EB1089 was significantly less calcemic than 1,25(OH)2D and did not induce severe weight loss. This is the first report of a vitamin D analogue that significantly inhibits prostate cancer metastasis in vivo and that does so without producing cachexia or unacceptable hypercalcemia.

Exploring new strategies for the management of acute coronary syndromes.

Patients who present today with an acute coronary syndrome face a substantially lower risk of death, recurrent myocardial infarction, or severe ischemia than patients did a decade ago. Researchers are pursuing new strategies to further improve the outcomes of patients with acute coronary syndromes. These strategies may be grouped into 3 paradigms: (1) restoration and maintenance of coronary flow at the site of culprit lesion; (2) reduction of infarct size, reperfusion injury, and postischemic dysfunction; (3) stabilization of the coronary arterial wall and its interaction with the bloodstream to reduce recurrent ischemic events. The last approach encompasses strategies to alter the underlying vascular pathophysiology that leads to plaque instability and coronary thrombosis. Investigation into each of these paradigms may yield new strategies that will be incorporated into standard clinical management of acute coronary syndromes in coming years. With so many mechanistically different approaches to the management of acute coronary syndromes, clinicians have reason for optimism that continued progress will further reduce the morbidity and mortality associated with acute coronary syndromes and the likelihood of their recurrence.

In vivo phosphorus-31 spectroscopic imaging in patients with global myocardial disease.

The goals of this study were to determine whether abnormalities in phosphorus metabolism could be noninvasively detected using phosphorus-31 nuclear magnetic resonance spectroscopy in patients with dilated cardiomyopathy and left ventricular hypertrophy, and whether these patient groups could be distinguished from each other based on parameters obtained using this technique. Seventeen patients and 14 control subjects were studied using nuclear magnetic resonance spectroscopy. Spectra were obtained from the human heart at rest using 3-dimensional spectroscopic imaging as a localization technique. Data were acquired over an average volume of 48 cc in 26.3 minutes using a 2 tesla imaging and spectroscopy unit. The ratio of phosphocreatine to adenosine triphosphate was 0.89 +/- 0.88 (mean +/- standard error) in normal subjects and did not differ significantly in patients with dilated cardiomyopathy or left ventricular hypertrophy. A prominent peak in the phosphodiester region was seen much more frequently in patients with dilated cardiomyopathy, resulting in significantly higher ratios of phosphodiester to phosphocreatine (1.28 +/- 0.35) and phosphodiester to adenosine triphosphate (0.79 +/- 0.18) in this group compared to normal subjects (0.33 +/- 0.08 and 0.29 +/- 0.08, respectively). However, the various patient groups could not be reliably distinguished from each other based on spectral patterns. These studies demonstrate the feasibility of performing phosphorus-31 nuclear magnetic resonance spectroscopic imaging in patients with myocardial disease. The initial results indicate that, under resting conditions, the ratio of phosphocreatine to adenosine triphosphate is not consistently altered in patients with severe global cardiomyopathies or hypertrophy. Phosphodiesters are elevated in some patients with dilated cardiomyopathy, a finding that may signify abnormal phospholipid metabolism in this condition.

Rationale and design of the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study that evaluates atorvastatin in unstable angina pectoris and in non-Q-wave acute myocardial infarction.

The goal of the Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) study is to determine whether early, rapid, and profound cholesterol lowering therapy with atorvastatin can reduce early recurrent ischemic events in patients with unstable angina or non-Q-wave acute myocardial infarction. Within 1 to 4 days of hospitalization for one of these conditions, 2,100 patients will be randomly assigned to receive atorvastatin, 80 mg/day, or placebo in a double-blind design. Both groups receive dietary counseling. Over a 16-week follow-up period, the primary outcome measure is the time to occurrence of an ischemic event, defined as death, nonfatal acute myocardial infarction, cardiac arrest with resuscitation, or recurrent symptomatic myocardial ischemia requiring emergency rehospitalization. Secondary outcome measures are the time to occurrence and incidence of each of the primary outcome components, as well as nonfatal stroke, worsening angina, congestive heart failure requiring hospitalization, and need for coronary revascularization not anticipated before randomization. The sample size of 1,050 patients in each group is expected to provide 95% power to detect a 30% reduction in the primary outcome measure with a 5% level of significance. The results of the MIRACL study will determine the utility of profound cholesterol lowering as an early intervention in acute coronary syndromes.

Vitamin D receptor content and transcriptional activity do not fully predict antiproliferative effects of vitamin D in human prostate cancer cell lines.

Prostate cancer cell lines exhibit variable growth suppression by the hormonal form of vitamin D3, 1,25-Dihydroxyvitamin D3 [1,25 (OH)2D] (1,25 D3). To understand the molecular basis for this differential sensitivity to 1,25 D3, we compared growth response to 1,25 d3, vitamin D receptor (VDR) content and VDR transcriptional activity in four well-characterized human prostate cancer cell lines: LNCaP, DU145, PC-3 and ALVA-31. In PC-3 and DU145 cells, relative lack of growth inhibition by 1,25 D3 (< 10% inhibition) correlates with very low levels of VDR (9-15 fmol/mg protein) compared to classical vitamin D3 target tissues (approximately 75-200 fmol/mg protein). Transfection of DU145 and PC-3 cells with a VDR cDNA expression vector is sufficient to establish growth sensitivity to 1,25 D3, suggesting that low VDR levels are responsible for the failure of these cell lines to respond to 1,24 D3. LNCaP cells are highly sensitive to growth inhibition by 1.25 D3 (approximately 55% inhibition) and contain approximately 2-3-fold more VDR (25 fmol/mg) than the relatively 1,25 D3-insensitive PC-3 and DU145 cell lines. However, ALVA-31 cells display less than 20% growth inhibition to 1.25 D3 although they contain the highest levels of VDR (45 fmol/mg) of the four cell lines. Thus, sensitivity to growth inhibition by 1,25 D3 does not correlate with VDR content in ALVA-31 and LNCaP cells. This lack of correlation between VDR density and growth responses to 1,25 D3 led us to investigate VDR-mediated gene transcription in these cell lines. We employed two different naturally occurring vitamin D response elements (VDREs) linked to a reporter gene. Reporter gene activation by 1,25 D3 correlated well with VDR content in all four cell lines. Therefore, compared to LNCaP cells, decreased sensitivity of ALVA-31 to growth inhibition by 1,25 D3 is not due to a decrease in the general transcriptional activity of VDR. We conclude that growth inhibition by 1,25 D3 in prostate cancer cells requires VDR but that this response is modulated by non-receptor factors that are cell line-specific.

Magnetic resonance spectroscopy. Evaluation of ischemic heart disease.

Magnetic resonance spectroscopy (MRS) is a valuable tool for the study of myocardial ischemia. Phosphorus (31P) MRS can detect changes in high-energy phosphates resulting from ischemia and has been used to determine the sensitivity of metabolic changes to ischemia as well as to investigate the metabolic factors important for myocardial dysfunction. The mechanisms mediating postischemic dysfunction have been investigated using 31P MRS, as have interventions to limit metabolic and functional damage from ischemia. These investigations have laid the groundwork for human cardiac studies. While abnormalities following myocardial infarction have been shown in man, further work must be performed to reliably acquire localized spectra under conditions of ischemia.