Modulation of Endothelial Cell Survival by an Inhibitor of Angiogenesis Thrombospondin-1: a Dynamic Balance

Angiogenesis is a process of capillary formation from pre-existing blood vessels. It is tightly controlled by the balance between positive and negative environmental signals – inducers and inhibitors of angiogenesis in such a way that predominance of inducers results in angiogenesis and predominance of inhibitors – in vascular quiescence. Here we discuss the ability of the angiogenic stimuli to promote survival and the pathways they may utilize. We also summarize information available on the signaling events elicited in the endothelial cells by a naturally occurring inhibitor of angiogenesis Thrombospondin-1 (TSP-1), that result in the endothelial cell apoptosis and inhibition of angiogenesis in vivo. This ability to cause programmed cell death in vascular endothelium is not unique to TSP-1. A substantial number of known angiogenesis inhibitors can also trigger apoptosis in the activated endothelial cells. This fact argues for the possibility of apoptosis to be a common denominator for a major fraction of anti-angiogenic molecules. If this is the case, it is equally possible that the ratio between environmental factors that control angiogenesis is interpreted within individual endothelial cell as a balance between pro-apoptotic and survival signals. Thus the relative strength of the death and survival signal or signals determines the fate of endothelial cell and therefore the fate of remodeling vessel.     

Inhibition of thyrotropin- and dibutyryl cyclic AMP-induced secretion of thyroxine and triiodothyronine by catecholamines.

Thyrotropin (TSH), 1 MU/ml and N6, O2'-dibutyryl adenosine 3',5-cyclic monophosphoric acid (dbcAMP) greatly enhanced the release of thyroxine (T4) and triiodothyronine (T3) from mouse thyroids incubated in vitro. L-Epinephrine (E) and L-norepinephrine (NE) strongly inhibited the TSH and dbcAMP-stimulated release of thyroid hormones; L-isoproterenol (IPNE) exerted a relatively weak inhibition. The inhibition by catecholamines was prevented by the alpha-adrenergic blocker, phentolamine; L-propranolol, a beta-adrenergic blocker, had no effect on the inhibition. The TSH-induced release of thyroid hormones was not affected by adrenergic blockers. Epinephrine did not affect the increase in thyroidal cAMP content induced by TSH. These results indicate that catecholamines act by way of an alpha-adrenergic receptor to suppress TSH-stimulated release of thyroid hormones at a point beyond cAMP formation.     

Iodoamino acid content and distribution in normal and abnormal human thyroids.

Iodoamino acid content and distribution were measured in adenomas, nodular goiters, thyroiditis and carcinomas and compared to thyroids from patients who died suddenly and normal thyroid tissues originally adjacent to diseased tissue. Tissues were hydrolyzed with Pronase, derivatized and analyzed by gas chromatography as had previously been reported for rat thyroids. Considerable overlap in values was found among adenomas, nodular goiters and papillary and follicular carcinomas as compared to values in normal thyroid tissue, but low values were also found in several diseased tissues. MIT/DIT and T3/T4 ratios were essentially constant in most tissues, even when T4 values were low. MIT/DIT ratios were, however, high and iodothyronines undetectable in all Hurthle cell neoplasms, clearly differentiating them from other thyroid conditions.     

Serum triiodothyronine concentration in the iodine-deficient rat

A low iodine diet fed to rats resulted in decreased absolute amounts of circulating triiodothyronine and protein-bound iodine (PBI), but an increase in the triiodothyronine: protein-bound iodine ratio. Since baseline protein-bound iodine values were high, it is possible the “normal” diet fed was in fact a high iodine one. In this event, the fall in protein-bound iodine with the low iodine diet would be greater than that in thyroxine concentration. The relative increase in triiodothyronine compared to PBI concentration with the low iodine diet could result from preforential synthesis and secretion of triiodothyronine by the iodine-deficient thyroid; or could be due to accelerated removal of thyroxine due to increased peripheral utilization. Either change could keep body metabolism normal or nearly so over an extended period of iodine want.     

The modulation of thrombospondin and other naturally occurring inhibitors of angiogenesis during tumor progression

Summary Fifteen different natural inhibitors of angiogenesis have now been identified that are produced by mammalian cells and are able to blockin vivo neovascularization. The majority of these are able to inhibit endothelial cell activitiesin vitro and all those tested have demonstrated significant antitumor activity. Most normal cells produce inhibitors of neovascularization that must be downregulated before the cells can develop into angiogenic, malignant tumors. In several cases the production of inhibitors ceases when tumor suppressor genes are inactivated. In the BT549 human breast carcinoma cell line, the reintroduction of a wild type p53 tumor suppressor gene resulted in the stimulation of the secretion of an inhibitor of angiogenesis, thrombospondin-1, and as a result the cells lost their angiogenic phenotype and became able to suppress angiogenesis induced by the parental tumor line. These results provide a new example of tumor suppressor gene control of a natural inhibitor of angiogenesis and add support to the concept that thrombospondin loss may play an important role in the development of some human breast cancers.     

Smad4/DPC4-mediated tumor suppression through suppression of angiogenesis

Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.     

Norepinephrine and thyrotropin effects on the thyroid in vitro: simultaneous stimulation of iodide organification and antagonism of thyroxine release

Norepinephrine (NE), which has previously been shown to inhibit TSH-induced T4 release by mouse thyroids in vitro, was found to stimulate iodide organification. The concentration of NE (6 X 10(-7) M) necessary to stimulate organification of iodide was 10 times less than the concentration (6 X 10(-6) M) required for inhibition of TSH-induced T4 release. Both actions of NE were exerted through an alpha-adrenergic receptor, since they were inhibited by phentolamine but not by l-propranolol. One milliunit of TSH maximally stimulated T4 release only, but larger amounts (100 mU) also stimulated organification. TSH stimulation of T4 release and organification was not affected by adrenergic antagonists and therefore was not mediated by adrenergic receptors. N6, O2-Dibutyryl cAMP and isobutylmethylxanthine, like TSH, stimulated T4 release. Their actions were inhibited by NE. However, both compounds, unlike TSH, failed to enhance organification in mouse thyroids. The effects of TSH and NE on the cAMP content of incubated mouse thyroids were also studied. TSH induced a prolonged increase in thyroidal cAMP during the 90-min incubation; this increase was unaffected by alpha- or beta-adrenergic antagonists. In contrast, NE (6 X 10(-5) M) produced a transient but significant increase in cAMP only within the first 5 min. Unlike the action of NE on organification, this short term stimulatory effect on cAMP production was mediated by a beta-adrenergic receptor, since it was blocked by l-propranolol but not by phentolamine. The following conclusions were reached: 1) stimulation of iodide organification and thyroid hormone release involves different sensitivity thresholds for TSH and NE; 2) TSH stimulation of iodide organification, hormone release, and cAMP formation is not exerted through adrenergic receptors; 3) NE stimulates organification and inhibits TSH-stimulated T4 release through alpha-adrenergic receptors, but stimulates cAMP production through beta-receptors; and 4) cAMP may not be the mediator of all TSH actions on the thyroid.