The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

Interstitial collagenase is required for angiogenesis in vitro.

Human umbilical vein endothelial cells (HUVECs) invade collagen gels and establish vascular-like structures within the gel following stimulation with phorbol esters. This process was quantitated by measuring release of radioactivity from gels composed of [3H]collagen. Collagen was steadily degraded over the period of several weeks by phorbol ester-treated cells while little collagenolysis by cells not receiving phorbol ester was noted. Examination of matrix metalloproteinases (MMPs) secreted by HUVECs revealed a prominent induction of interstitial collagenase. Production of the mature forms of gelatinase A was also stimulated, as was the secretion of gelatinase B. Stromelysin was not detected. Two inhibitors of MMPs, the naturally occurring tissue inhibitor of metalloproteinases (TIMP; 10 micrograms/ml) and the synthetic, peptide inhibitor BB-94 (1 microM) were both effective at blocking HUVEC-mediated collagen degradation. Morphological examination of control, PMA-treated HUVECs, as well as PMA-treated HUVECs receiving TIMP or BB-94, revealed that MMP inhibition resulted in a block to invasion and tubule formation within the collagen gels. Similar results for MMP expression and inhibition of tubule formation in vitro were obtained with human dermal microvascular endothelial cells. Examination of collagen proteolytic fragments revealed that both BB-94 and TIMP blocked cleavage of the alpha 1 and alpha 2 chains of type I collagen and the appearance of tropocollagen fragments A and B, demonstrating that the inhibitors were acting directly upon interstitial collagenase. Our results demonstrate that interstitial collagenase is required for angiogenesis in vitro.

A potential role for N-cadherin in mediating endothelial cell-smooth muscle cell interactions in the rat vasculature.

BACKGROUND: The cadherins are a family of calcium-dependent cell adhesion molecules that play a critical role in morphogenetic cell interactions in the embryo. N-cadherin has been identified in neural and non-neural tissues, including developing muscle; however, there are few reports of N-cadherin expression in adult mammals. We attempted to identify N-cadherin in the rat vasculature and to ask whether it has a role in mediating vascular cell interactions. EXPERIMENTAL DESIGN: Vascular tissues were isolated from Fischer rats and analyzed immunohistochemically and by Western blotting using pan-cadherin and N-cadherin antibodies. Anti-N-cadherin antibodies were used in an in vitro cell adhesion assay to determine if N-cadherin mediates interactions between smooth muscle cells (SMC) and endothelial cells (EC). RESULTS: Western blot analysis of vascular tissues reveals a 135 kilodalton (kd) band that is recognized by anti-N-cadherin antibodies. N-cadherin localizes in the rat aorta to the endothelium and to the smooth muscle cell layer beneath the internal elastic membrane. A cell adhesion assay demonstrates that rat SMCs and ECs adhere to each other in a Ca(2+)-dependent manner, and an anti-N-cadherin antibody inhibits approximately 25% of this adhesion. CONCLUSIONS: Our data indicate that N-cadherin is expressed in large vessels of the rat and is capable of mediating EC-SMC adhesion in vitro. It is noteworthy that N-cadherin localizes to a population of SMCs in the rat aorta adjacent to ECs suggesting that N-cadherin may mediate EC-SMC interactions in vivo.

Placental and fetal development in straightbred and reciprocal crosses of Yorkshire and Ossabaw swine.

Placental and fetal development were compared in 7 Yorkshire (Y) and 12 feral, Ossabaw (F) gilts mated to Y and F boars to produce straightbred and reciprocal cross litters (YY, YF, FY and FF, sire listed first). Gilts were slaughtered at d 75 +/- 2 of gestation. Ovulatory rate and litter size were higher in Y than in F gilts; however, rates of embyonal survival were similar in all groups. Groups differed in fetal body weight, metabolic body weight, crown-to-rump length, placental weight, metabolic placental weight and placental surface area, respectively. The YY group had highest values and the FF group smallest values for all variables except placental surface area. Pearson product-moment correlation coefficients of placental development with fetal length and weight across breeding groups were significant (e.g., placental surface area: fetal length, r = .67; placental surface area: fetal metabolic body weight, r = .76). It is concluded that the relationship between placental and fetal development observed in domestic swine is present in feral swine and crosses between domestic and feral swine at this gestational stage. These developmental measures are significantly influenced by fetal genotype and uterine environment but also by the breed of sire.