Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization - PubMed (original) (raw)

Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization

A Pozzi et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Integrin alpha1beta1 is a collagen receptor abundantly expressed on microvascular endothelial cells. As well as being the only collagen receptor able to activate the Ras/Shc/mitogen-activated protein kinase pathway promoting fibroblast cell proliferation, it also acts to inhibit collagen and metalloproteinase (MMP) synthesis. We have observed that in integrin alpha1-null mice synthesis of MMP7 and MMP9 was markedly increased compared with that of their wild-type counterparts. As MMP7 and MMP9 have been shown to generate angiostatin from circulating plasminogen, and angiostatin acts as a potent inhibitor of endothelial cell proliferation, we determined whether tumor vascularization was altered in the alpha1-null mice. Tumors implanted into alpha1-null mice showed markedly decreased vascularization, with a reduction in capillary number and size, which was accompanied by an increase in plasma levels of angiostatin due to the action of MMP7 and MMP9 on circulating plasminogen. In vitro analysis of alpha1-null endothelial cells revealed a marked reduction of their proliferation on both integrin alpha1-dependent (collagenous) and independent (noncollagenous) substrata. This reduction was prevented by culturing alpha1-null cells with plasma derived from plasminogen-null animals, thus omitting the source from which to generate angiostatin. Plasma from tumor-bearing alpha1-null animals uniquely inhibited endothelial cell growth, and this inhibition was relieved by the coaddition of either MMP inhibitors, or antibody to angiostatin. Integrin alpha1-deficient mice thus provide a genetically characterized model for enhanced angiostatin production and serve to reveal an unwanted potential side effect of MMP inhibition, increased tumor angiogenesis.

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Figures

Figure 1

Figure 1

Morphological analysis of tumor-bearing wild-type (WT) and α1-null mice. (A and B) Typical gross appearance of tumors grown from four quadrant bolus injections of 105 cells in the subcutis of wild-type and α1-null animals. (A) Complete dissection of dorsal skin after 14 days. (B) Close-up of tumors after 10 days (scale bar = 1 mm). In the wild-type host, tumors appeared strikingly more vascular than those grown in α1-null mice. This difference was apparent regardless of the size of the tumors. (C) Blood vessel staining of tumor sections from wild-type and α1-null mice showing a reduction in size and number in the latter. (Objective magnification: ×20.)

Figure 2

Figure 2

(A) Tumor volume in wild-type (WT) and α1-null (α1-KO) hosts at 14 days. ○ indicate the total tumor volume for each mouse and the solid line the mean of samples. Four independent experiments were performed, each with a minimum of three animals per genotype. α1-null hosts show a significant reduction in tumor volume (P < 0.05) (B) Proportion of tumors growing from the initiating cell bolus after 10–14 days. The number of tumors grown from the injected initiating cell bolus of 105 cells was counted for each animal. ○ indicate the mean result for each experiment, and the solid line the mean of means. Seven independent experiments were performed, each with a minimum of three animals per genotype. α1-null hosts show a significant reduction in tumor take (P < 0.05). Although tumor size increased with growth time, percentage take did not change with growth time. (C) Tumors from α1-null hosts show reduced vascularity as measured by extent of CD31 positive structures (P < 0.05). Bars and errors indicate the mean and SD (five tumors per genotype were examined in experiment 1, three per genotype in experiment 2, seven per genotype in experiment 3, five per genotype in experiment 4). (D) α1-Null primary lung endothelial cells show reduced proliferation on collagen and fibrinogen as compared with wild-type cella (P < 0.05). The y axis indicates absolute cpm incorporation of primary endothelial cells grown as described in Materials and Methods. Bars and errors indicate the mean and SD of three different experiments (with a total of six animals for each group). (E) Inhibition of proliferation of α1-null endothelial cells is the result of a soluble factor. The y axis is the same as in D. Means and SD are of triplicate samples of endothelial cells from three wild-type and α1-deficient mice. α1-Null cells grow less than their wild-type counterparts when cultured in complete medium (“clonetics”). Changing the medium at 12 hourly intervals (“change”) rescues α1-null cell growth, returning growth to wild-type levels; while adding conditioned media from α1-null cells (“swap”) prevents wild-type cell growth. (F) Immunoprecipitation from lysates of wild-type (W) and α1-null (K) endothelial cells with anti-β1 or anti-αv antibody. The designation α2-v indicates the positions of subunits α2, 3, 4, 5, and v, which are incompletely resolved from one another. Anti-β1 antibody failed to precipitate α1 integrin in α1-null endothelial cells (Left). A light exposure of the same membrane is shown to reveal no differences in α2-v bands between wild-type and α1-null endothelial cells (Middle). Anti-αv antibody failed to precipitate αv integrin in lysates of endothelial cells of both genotype, unlike in those of control embryonic fibroblasts (Ct) (Right).

Figure 3

Figure 3

MMP synthesis in wild-type (WT) and α1-null mice. (A) Gelatin and casein zymograms of conditioned media from full thickness skin explants from two wild-type and two α1-null mice. Increased levels of MMP2 (72- to 74-kDa pair), MMP9 (92- to 94-kDa pair), and MMP7 (21–28 kDa) are seen in the two α1-null samples as compared with the wild type. (B) Gelatin and casein zymograms of conditioned medium from wild-type and α1-null primary endothelial cells showing increased level of MMP9 and MMP7 in the α1-null cells. (C) Gelatin zymogram of gelfoam sponges from wild-type and α1-null mice. In most samples, MMP9 levels are increased in α1-null samples relative to the wild-type samples. (D) Gelatin zymogram of plasma from tumor-bearing wild-type and α1-null mice showing similarly increased levels of MMP9 in the plasma from α1-null samples. MMP9 bands were quantified by densitometry and compared with the measured tumor volume (cm3) for each animal. There is a striking inverse correlation between MMP9 expression and tumor size within the two groups.

Figure 4

Figure 4

(A) Angiostatin synthesis in wild-type and α1-null mice. (Upper) Western blot of plasma angiostatin. Increased levels of angiostatin (40- to 45-kDa fragments of plasminogen) are evident in the plasma of α1-null mice (K) with tumors or sponge implants as compared with their wild-type (W) counterparts. Membranes incubated with secondary antibody alone or irrelevant IgG were used as negative controls (not shown). (_Lowe_r) Portion of a Coomassie staining of an equal loading of the plasma used for Western blot analysis, showing integrity of the samples. (B) Plasma from tumor-bearing α1-null mice generates angiostatin from mouse serum. Two percent mouse serum (S) was incubated with 7.5 μg total plasma proteins from tumor-bearing wild-type (WT) or α1-null (α1-KO) mice. This amount of total proteins did not yield visible angiostatin bands in plasma from tumor-bearing wild-type (WT −S) and α1-null (α1-KO −S) mice incubated in absence of mouse plasma. In contrast, angiostatin fragments were detected in mouse serum incubated with plasma from tumor-bearing α1-null mice (α1-KO +S), but not in the sample incubated with plasma from tumor-bearing wild-type mice (WT +S). (C) BB94 prevents the generation of angiostatin from mouse serum. Two percent mouse serum was incubated with 7.5 μg total plasma proteins from tumor-bearing α1-null mice in the presence or absence of 2.5 μM BB94. The presence of 2.5 μM BB94 (+ BB94) prevents angiostatin formation.

Figure 5

Figure 5

(A) Effect of plasma from untreated and tumor-bearing mice on HUVEC proliferation. The y axis indicates absolute incorporation of 3H-thymidine. Means and SDs are of triplicate samples of plasma from three untreated or tumor-bearing animals of each genotype. Only plasma from tumor-bearing α1-null mice reduces HUVEC proliferation (P < 0.05). (B) BAEC proliferation in the presence or absence of antiangiostatin. Angiostatin inhibits BAEC proliferation (− anti-K1–3), whereas addition of anti-K1–3 rescues their proliferation (+ anti-K1–3) (P < 0.05). (C) Angiostatin is responsible for the growth inhibition of α1-null cells. (Left) Cell growth in endothelial growth medium containing 5% FCS (clonetics). Addition of antiangiostatin antibody (Anti-K1–3) at 20 μg/ml rescues growth of α1-null cells. (Right) Growth in media containing 5% mouse plasma instead of FCS. Growth of α1-null cells is inhibited in presence of normal mouse plasma (WT plasma), but is not inhibited in the presence of plasma from plasminogen null mice (plg-null plasma). Means and SDs are of triplicate samples of endothelial cells from three wild-type and α1-null mice. (D) Effect of coaddition of antiangiostatin or TIMP-1 on HUVEC proliferation in the presence of plasma from tumor-bearing α1-null mice. The y axis is as in A. HUVEC were grown for 96 hr with plasma from tumor-bearing α1-null mice (1 mg/ml) in the presence of anti-K1–3 or TIMP-1 at concentrations indicated. Treatment with either antibody or TIMP-1 rescued HUVEC proliferation (P < 0.05). (E) Effects of addition of BB94, at concentrations indicated, on BAEC proliferation in the presence of 1 mg/ml plasma from tumor-bearing wild-type or α1-null mice. The y axis is as in A. Inhibition of BAEC growth by plasma from α1-null tumor-bearing mice is rescued by BB94 (P < 0.05).

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