Jelly belly: a Drosophila LDL receptor repeat-containing signal required for mesoderm migration and differentiation.

Inductive interactions subdivide the Drosophila mesoderm into visceral, somatic, and heart muscle precursors. The muscle precursors form organs by executing tissue-specific migrations and cell fusions. We identified a novel gene, jelly belly (jeb), which is required for visceral mesoderm development. jeb encodes a secreted protein that contains an LDL receptor repeat. In jeb mutants, visceral mesoderm precursors form, but they fail to migrate or differentiate normally; no visceral muscles develop. Jeb protein is produced in somatic muscle precursors and taken up by visceral muscle precursors. jeb reveals a signaling process in which somatic muscle precursors support the proper migration and differentiation of visceral muscle cells. Later in embryogenesis, jeb is transcribed in neurons and Jeb protein is found in axons.

Stimulation of bone resorption results in a selective increase in the growth rate of spontaneously metastatic Walker 256 cancer cells in bone.

To test the hypothesis that bone metastasis is related to the rate of bone remodeling, we have examined the effect of enhanced bone resorption on the growth of spontaneously metastatic Walker 256 (W256) cancer cells. Bone resorption was stimulated in male Fischer rats by injecting Rice H-500 Leydig tumor cells subcutaneously. The resorptive response of the skeleton was confirmed in a pilot study by evaluating parameters of bone morphometry after 4, 7 and 10 days of tumor burden. The distal femoral epiphyses had 35 +/- 10% more osteoclast surface, 83 +/- 11% less osteoblast surface, and 46 +/- 5% less trabecular bone after 10 days of tumor burden, compared to non-tumor-bearing controls. To evaluate the effect of Leydig tumor-induced bone resorption on the growth response of W256 cells, 20 rats were injected intramuscularly with 2 x 10(7) W256 cells, and 20 rats were vehicle-injected. Two days later, 10 rats from each group were injected subcutaneously with Leydig tumor cells. Twelve days after W256/vehicle injection, rats were injected with [3H]thymidine, killed 2 h later, and their femurs, liver, lungs and kidneys were processed for histology. In rats injected with Leydig tumor cells only, enhanced bone resorption was confirmed by a 40 +/- 4% increase in serum calcium concentration, a 48 +/- 8% decrease in trabecular bone content, and a 72 +/- 15% decrease in osteoblast surface, compared with non-tumor-bearing rats. Metastatic W256 cells adjacent to trabecular bone in Leydig tumor-bearing rats had a 56 +/- 18% greater relative [3H]thymidine labeling index (TdR) than did W256 cells in the bones of non-Leydig tumor-bearing rats. The TdRs of W256 cells in the liver, lungs, and kidneys were not affected by Leydig tumor burden. In this model, enhanced bone resorption was associated with the selective growth promotion of metastatic W256 cells in bone, suggesting the existence of a bone-derived factor which is mitogenic to W256 cells.

A quantitative model for spontaneous bone metastasis: evidence for a mitogenic effect of bone on Walker 256 cancer cells.

A new model for the study of spontaneous bone metastasis has been developed which allows for the quantification of metastatic tumor burden and cancer cell growth rate, and which describes the progressive changes in bone morphology. Walker 256 (W256) cells or vehicle were injected into the left upper thigh muscle of male Fischer rats, which were killed 7, 10 or 14 days later. By day 7, metastases had appeared in the distal femur, in the glomeruli of the kidney, and diffusely throughout the liver and lungs. The extent of tumor burden in these organs increased over time. In the femur, 14 days of tumor burden was associated with a 53 +/- 10% decrease in trabecular bone content, a 61 +/- 15% increase in osteoclast surface, and a 95 +/- 10% decrease in osteoblast surface, as compared with non-tumor-bearing controls. By autoradiography, metastatic tumor cells in all organs were determined to have greater growth rates than did cells in the primary tumor. However, within the femur, W256 cells located adjacent to trabecular bone surfaces had a 33 +/- 7% greater growth rate than did W256 cells located > 50 microns from bone surfaces (P < 0.05), suggesting a mitogenic effect of bone.

Transferrin glycosylation in hypoxia.

The aim of this study was to examine the effect of reduced O2 tension on the glycosylation of transferrin. Rats were placed in a hypobaric chamber (380 mmHg) that corresponded to an altitude of 5486 m above sea level for 21 days. The animals responded with marked increases in hematocrit (from 44 to 76%) and cardiac weight, and with reductions in the concentration of plasma transferrin averaging 15%. Analyses of their plasma transferrin by serial anion-exchange and lectin affinity chromatography revealed no changes in the extent of glycan branching. However, there was a moderate rise in the proportion of fucosylated transferrin molecules (fucosylation index) and a slight decrease in the transferrin fraction bearing a tetrasialylated biantennary glycan. The fucosylation index correlated positively with plasma transferrin concentrations in the test animals, but not in the controls. In contradistinction to the situation with transferrin, hypoxic rats exhibited a reduced fucosylation index of immunoglobulin G.

Enhanced cancer metastasis after monocrotaline-induced lung injury.

The lung is a target in several models of environmentally induced injury and is also a common site for the growth of metastases from circulating cancer cells. In these experiments, we have tested the hypothesis that pulmonary damage can facilitate the metastasis of cancer to the lung. We have studied the effect of monocrotaline-induced lung injury on the retention and metastasis of intravenously injected Walker carcinosarcoma 256 cells in the lung and the effect of this injury on spontaneous metastasis in animals with intramuscular tumor transplants. Female Wistar rats were given a single subcutaneous injection of monocrotaline (60 mg/kg). The degree of lung injury after monocrotaline was assessed by bronchoalveolar lavage, by histological and ultrastructural examination, and by measurement of right ventricular hypertrophy. To assess the effects of monocrotaline on metastasis, animals were injected iv with 2 X 10(7) [125I]iododeoxyuridine-labeled or unlabeled Walker 256 carcinosarcoma cells at various periods of time (1 day to 20 days) after monocrotaline. The retention of labeled cells was determined by gamma counts of lungs 24 hr after injection. There was a direct correlation between the severity of lung injury and the number of cancer cells retained in the lung 24 hr after injection. Metastasis was quantified by morphometric analysis of histologic sections prepared from lungs 1 week after an injection of unlabeled cells. The median area of lung involved by tumor after iv injection was 39% for rats injected with cancer cells 10 days after monocrotaline vs 3% for controls. In studies on spontaneous metastasis, rats were given an intramuscular injection of Walker 256 cells 5 days after monocrotaline and metastasis was quantified by morphometry 7 days after tumor transplantation. The median tumor burden of animals pretreated with monocrotaline was 37% vs 8% for controls. We conclude that lung injury initiated by monocrotaline can facilitate the spread of the rat Walker 256 carcinosarcoma.

Lung angiotensin converting enzyme activity in chronically hypoxic rats.

A study was carried out to test the hypothesis that the reduced lung angiotensin converting enzyme (ACE) activity which occurs in chronic hypoxia is related to the development of pulmonary hypertension rather than to hypoxia per se. Right ventricular mean systolic pressure (Prvs, mm Hg) and ACE activity (nmol/mg protein/min) in lung tissue homogenates were measured in seven groups of four rats placed in a hypobaric chamber (380 mm Hg; 51 kPa) for two to 24 days. Identical measurements were made on 11 groups of four rats, which were placed in the chamber for 24 days and then allowed to recover in room air for one to 153 days. After two days of hypoxia the mean Prvs (25.5 (SD 3.7] and the mean lung ACE activity (56 (4.6] did not differ significantly from control values. Exposure to hypoxia for four to 24 days caused a progressive increase in mean Prvs to 44.4 (5.9) and a progressive reduction in mean lung ACE activity to 34 (4.0). During recovery lung ACE activity increased and Prvs decreased, so that normal values were achieved by 15 and 56 days respectively. Decreased lung ACE activity may be related to haemodynamic factors associated with pulmonary hypertension rather than to hypoxia.

Pulmonary hypertension induced in rats by monocrotaline and chronic hypoxia is reduced by p-chlorophenylalanine.

We have studied the role of 5-hydroxytryptamine (5-HT) in monocrotaline pulmonary hypertension and chronic hypoxic pulmonary hypertension in rats using p-chlorophenylalanine (PCPA) which inhibits 5-HT synthesis. We measured right ventricular mean systolic pressure (Prvs), right ventricular hypertrophy, medial thickness of muscular pulmonary arteries, and muscularization of pulmonary arterioles 17 days after a single dose of monocrotaline (60 mg/kg) and after 26 days of chronic hypobaric hypoxia (380 mm Hg). In monocrotaline pulmonary hypertension, pretreatment with PCPA (500 mg/kg) was associated with significant reductions (p less than 0.05) in Prvs (29%), right ventricular hypertrophy (33%), and medial thickness of muscular pulmonary arteries (14%). In chronic hypoxic pulmonary hypertension, pretreatment with PCPA was associated with significant reductions in Prvs (20%), right ventricular hypertrophy (28%), medial thickness of muscular pulmonary arteries (14%), and muscularization of pulmonary arterioles (47%). 5-HT may play a role in the development of monocrotaline pulmonary hypertension and chronic hypoxic pulmonary hypertension in rats.

Failure to show decrease in small pulmonary blood vessels in rats with experimental pulmonary hypertension.

We induced chronic pulmonary hypertension in one group of rats by exposing them to chronic hypobaric hypoxia (380 mm Hg for three weeks) and in another group by administering a single subcutaneous dose of monocrotaline (60 mg/kg body weight). Both groups of rats showed increase of the right ventricular mean systolic blood pressure and right ventricular hypertrophy. We measured the surface area of histological sections of the left or right lungs and counted all small blood vessels with an external diameter of less than 50 microns and with a definite elastic coat lying distal to respiratory bronchioles. In the 10 rats with chronic hypoxic pulmonary hypertension the mean total number of small pulmonary blood vessels was 428.8 +/- 96.9 (SD) compared with 337.8 +/- 91.9 in 10 untreated control rats. The number of small pulmonary blood vessels per mm2 of lung tissue was 4.0 +/- 1.3 in the chronically hypoxic rats compared with 3.8 +/- 1.2 in the controls. The mean total number of small pulmonary blood vessels in nine rats with monocrotaline-induced pulmonary hypertension was 396.8 +/- 61.7 compared with 384 +/- 55.4 in three control rats. The number of small pulmonary blood vessels per mm2 lung tissue was 3.3 +/- 0.6 in the rats treated with monocrotaline compared with 3.6 +/- 0.6 in the control group. We conclude that the number of small pulmonary blood vessels is not reduced in rats with pulmonary hypertension induced by chronic hypoxia or monocrotaline.

Lung angiotensin converting enzyme activity in rats with pulmonary hypertension.

We have studied serum and lung tissue angiotensin converting enzyme (ACE) activity in female Wistar rats with pulmonary hypertension induced by two different methods. Chronic pulmonary hypertension was produced in one group of 10 rats (CH) by confinement in a hypobaric chamber (380 mmHg) for three weeks, and in another group fo 10 rats (M) by a single subcutaneous injection of monocrotaline (60 mg/kg body weight). In these two groups of tests rats and in 20 untreated controls (C), we evaluated right ventricular mean systolic blood pressure (Prvs mmHg), right ventricular hypertrophy, and serum ACE (n mol/ml/min). In lung tissue homogenate, we measured the specific activity of ACE (n mol/mg protein/min), alkaline phosphatase (AP) (IU/mg protein) and lactic dehydrogenase (LDH) (IU/mg protein). The Prvs in groups, C, CH, and M was 25 +/- 7 SD, 41 +/- 7, and 51 +/- 5, respectively. The ratio of right ot left ventricular weight (RV/(LV + S)%) in groups, C, CH, and M was 29 +/- 4, 52 +/- 5, and 56 +/- 7, respectively. The lung tissue ACE in groups C, CH, and M was 85 +/- 11, 65 +/- 20, and 22 +/- 5, respectively. In groups CH, and M the Prvs and RV/(LV + S)% were significantly elevated above control values while lung ACE was significant decreased (p less than 0.05). There was a significant inverse relationship between lung ACE on one hand, and Prvs (r = - 0.73) and RV/(LV + S)% (r = - 0.71) on the other hand. Serum ACE and lung AP were unchanged. In group M there was a slight but significant reduction in lung LDH. Chronic pulmonary hypertension, irrespective of its method of production, is associated with decreased lung ACE. The reduction in lung ACE is inversely proportional to the severity of pulmonary hypertension and right ventricular hypertrophy.

Angiotensin converting enzyme activity and evolution of pulmonary vascular disease in rats with monocrotaline pulmonary hypertension.

We have investigated the role of angiotensin converting enzyme (ACE) in the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular disease in rats given a single subcutaneous injection of the pyrrolizidine alkaloid monocrotaline. Thirty-six young female Wistar rats were divided into a test group of 27 animals and a control group of nine animals. Each test rat was given a single subcutaneous injection of monocrotaline (60 mg/kg body weight). On the first, third, fifth, seventh, tenth, twelfth, fourteenth, seventeenth, and twenty-second days after the injection of monocrotaline the mean right ventricular systolic blood pressure was measured in one control and three test rats. The animals were then killed and we measured the specific activity of ACE in serum and lung homogenate. We also evaluated muscularisation of pulmonary arterioles, medial hypertrophy of muscular pulmonary arteries, and right ventricular hypertrophy. The sequence of changes was as follows: muscularisation of pulmonary arterioles and medial hypertrophy of muscular pulmonary arteries were apparent seven days after administration of monocrotaline; pulmonary hypertension and reduced lung ACE activity occurred after 10 days; right ventricular hypertrophy was detected after 12 days. Serum ACE activity was unchanged. It is concluded that the reduction in lung ACE activity is a result rather than a cause of the pulmonary hypertension. This reduction in lung ACE activity may be a protective mechanism designed to limit the elevation of the pulmonary arterial pressure.

Effect of intermittent normoxia on muscularization of pulmonary arterioles induced by chronic hypoxia in rats.

We studied the effect of continuous and intermittent normoxia for 6 and 20 wk on the muscularization of pulmonary arterioles in rats with chronic hypoxic hypertension. After 4 wk in a hypobaric chamber (380 mm Hg) the proportion of small pulmonary blood vessels with 2 elastic laminae (PVTEL) was 21.57 +/- 14.86% (SD) (n = 10) compared with 3.66 +/- 1.86% in 10 untreated control animals. Recovery using continuous normoxia and intermittent normoxia 16 h/day for 6 wk caused a reduction in PVTEL to 8.45 +/- 4.09% (n = 6) and 7.16 +/- 6.96% (n = 6), respectively. Right ventricular hypertrophy (RVH) was reversed by recovery using continuous normoxia for 6 wk but was unaffected by intermittent normoxia (16 h/day). Intermittent normoxia 8 h/day for 6 wk did not reduce the PVTEL or RVH. Continuous normoxia for 20 wk reversed the muscularization of small pulmonary vessels (PVTEL, 3.86 +/- 3.57%; n = 4) and RVH. Intermittent normoxia (16 h/day) for 20 wk significantly diminished the PVTEL to 7.39 +/- 3.73% (n = 5) but did not reduce RVH. Prolonged continuous normoxia slowly reversed the pulmonary hypertension, RVH, pulmonary vascular lesions, and polycythemia induced by chronic hypoxia. Intermittent normoxia (16 h/day) diminished the pulmonary vascular lesions but not the pulmonary hypertension, RVH, and polycythemia. Intermittent normoxia (8 h/day) was ineffective.

Mast cell stabilizing compound FPL 55618 reduces right ventricular hypertrophy and lung mast cell hyperplasia in chronically hypoxic rats.

Rats treated with chronic hypobaric hypoxia (21 days, 380 Torr) and mast cell stabilizing compound FPL 55618 had significantly less right ventricular hypertrophy and lung mast cell hyperplasia than rats subjected to chronic hypoxia alone. Right ventricular blood pressure was not reduced.