Direct test of potential roles of EIIIA and EIIIB alternatively spliced segments of fibronectin in physiological and tumor angiogenesis

Abstract

Fibronectin splice variants containing the EIIIA and/or EIIIB exons are prominently expressed in the vasculature of a variety of human tumors but not in normal adult tissues. To understand the functions of these splice variants in physiological and tumor angiogenesis, we used EIIIB-null and EIIIA-null strains of mice to examine neovascularization of mouse retinas, pancreatic tumors in Rip-Tag transgenic mice, and transplanted melanomas. Contrary to expectations, physiological and tumor angiogenesis was not significantly affected by the absence of either EIIIA or EIIIB splice variants. Tumor growth was also not affected. In addition, the expression levels of smooth muscle alpha actin, believed to be modulated by EIIIA-containing fibronectins, were not affected either. Our experiments show that despite their tight regulation during angiogenesis, the presence of EIIIA or EIIIB splice variants individually is not essential for neovascularization.

FIG. 1.

Expression of EIIIA- and EIIIB-containing FN. (A and B) RT-PCR performed with RNA isolated from heterozygous and null embryos. Lanes with asterisks contain samples taken from heterozygous animals (determined by genomic PCR). RT-PCR performed on mRNA isolated from EIIIA-and EIIIB-null animals shows the absence of these alternatively spliced exons. The positions of molecular size standards (in base pairs) are indicated to the left of the gels. (C) EIIIB-null tumors do not express EIIIB⁺ proteins. The arrow points to a vessel showing nonspecific staining of red blood cells (45). (D) A tumor section from EIIIB^+/− animal stained with antibody to EIIIB showing abundant vascular staining. Bars, 10 μm.

FIG. 2.

Physiological blood vessel formation in EIIIA- and EIIIB-null animals. (A) Retinal angiogenesis. Blood vessels, visualized with FITC-dextran, develop normally in EIIIA-null and EIIIB-null animals (right panels) compared with heterozygous littermate controls (left panels). Magnification, ×50. (B) Quantification of vessel density (per 100 pixels). Each data point represents vessel density measured in a 50,000- to 100-thousand pixel area. The difference in the median vessel density between EIIIA and EIIIB strains is probably due to mouse strain-specific factors (see Results). Four retinas were analyzed per genotype, and at least three areas per retina were quantified. See Materials and Methods for the description of the box plot.

FIG. 3.

Tail cross sections from 7-day-old mice were stained with hematoxylin and eosin. Tail sections from EIIIA^+/− (panel 1) and EIIIB^+/− (panel 3) and EIIIA^−/− (panel 2) and EIIIB^−/− (panel 4) mice are shown. Arrows point to arteries, and arrowheads point to veins. Bars, 10 μm.

FIG. 4.

Tumor blood vessels markedly upregulate FN and αSMA. (A) Normal pancreatic islet (bracket) surrounded by exocrine tissue. Cells were stained to detect FN (red) and αSMA (green), and the overlap between the two is shown in yellow. Note that in normal tissue, expression of αSMA could be found only around arterioles (arrowhead). (B) A tumor section from a Rip-Tag transgenic mouse, wild type for FN. Both FN and αSMA are highly upregulated around the tumor vasculature. Bars, 10 μm.

FIG. 5.

Expression of FN and splice variants in tumor blood vessels. In all panels, endothelial cells were stained to detect PECAM1 (red), smooth muscle cells are stained to detect αSMA (green), and NG2 (blue in panels A and B) or FNs (blue in panels C and D). (A) Wild-type pancreatic tissue. αSMA expression (green) is seen only around arterioles (arrowheads) near normal pancreatic islets. Small blood vessels express NG2 (blue) but not αSMA (green). (B to D) Rip-Tag tumors. Tumor blood vessels upregulate NG2 (blue) (B) and FN splice variantsV95 (blue) (C) and EIIIB (blue) (D), as well as αSMA (green) (B to D). Bars, 10 μm.

FIG. 6.

Islet tumorigenesis in the absence of EIIIB- or EIIIA-FN. (A) EIIIB^−/− and EIIIB^+/− Rip-Tag transgenic mice. The numbers of angiogenic islets isolated from 12-week-old Rip-Tag transgenic mice are shown (n is the number of animals used). (B) Tumor burden at 12 weeks of age. Tumor burden per mouse is the sum of volumes of all tumors in a mouse. (C) EIIIA^−/− and EIIIA^−/+ Rip-Tag transgenic mice. The numbers of angiogenic islets isolated from 11-week-old transgenic mice are shown. (D) Tumor burden at 11 weeks of age. Tumor burden was also assessed in 13-week-old animals on the mixed background, as well as in animals bred onto the BALB/c genetic background. Three independent experiments showed no statistically significant differences in the extent of the tumor growth between EIIIA^−/− and EIIIA^+/− animals (data not shown). Data from one experiment are shown. In this figure, P is the probability that the null hypothesis is true, as calculated by a two-tailed Student's t test. P values calculated according to the Wilcoxon sum-of-ranks test were 0.15, 0.12, 0.93, and 0.35 for panels A, B, C, and D, respectively. See Materials and Methods for information on the method used to generate the box plot.

FIG. 7.

Quantification of mRNA levels of FN splice variants in islet tumors isolated from EIIIA- or EIIIB-null animals. (A) RNase protection analysis of EIIIA-null and heterozygous tumors from Rip-Tag animals. Lanes 1 and 4 show the bands protected with the αSMA probe, lanes 2 and 5 show the bands protected with the V120 and EIIIB probes, and lanes 3 and 6 show the bands protected with the EIIIA probe. Results of quantification relative to the intensity of β-actin band are presented in Table 1. (B) RNase protection analysis of EIIIB-null and heterozygous tumors. Lane 1 contains undigested probes (V120, EIIIB, EIIIA, and αSMA). These four probes were used simultaneously for RNase protection. Lane 2 shows control hybridization using Saccharomyces cerevisiae RNA. In samples from EIIIB^+/− and EIIIB^−/− tumors, arrows point to V120-, V95-, EIIIB-, EIIIA-, and αSMA-protected bands. Results of quantification relative to the intensity of β-actin band are presented in Table 1.

FIG. 8.

Tumor vessel pericytes in EIIIA- and EIIIB-null Rip-Tag animals express αSMA. Frozen tumor sections were stained with antibodies to PECAM1 (red) and αSMA (green), and the overlap between the two is shown in yellow. (A) EIIIA-null tumor section. (B) EIIIB-null tumor section. Bars, 10 μm.

FIG. 9.

Growth of subcutaneous B16 tumors in EIIIA- and EIIIB-null mice. B16 F₀ tumor cells were injected subcutaneously into EIIIA-null or EIIIB-null or heterozygous mice at 8 weeks of age. Tumors were collected after 14 (EIIIA) or 17 (EIIIB) days. Medians (solid horizontal lines) and means (double lines) (EIIIB) are indicated. P values were calculated by the two-tailed Student's t test. Histological sections also showed no visible differences in tumors grown in null and heterozygous animals (data not shown).