Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment

Abstract

The endothelium is morphologically and functionally adapted to meet the unique demands of the underlying tissue. At the present time, little is known about the molecular basis of endothelial cell diversity. As one approach to this problem, we have chosen to study the mechanisms that govern differential expression of the endothelial cell-restricted von Willebrand factor (vWF) gene. Transgenic mice were generated with a fragment of the vWF gene containing 2,182 bp of 5' flanking sequence, the first exon and first intron coupled to the LacZ reporter gene. In multiple independent lines of mice, beta-galactosidase expression was detected within endothelial cells in the brain, heart, and skeletal muscle. In isogeneic transplantation models, LacZ expression in host-derived auricular blood vessels was specifically induced by the microenvironment of the heart. In in vitro coculture assays, expression of both the transgene and the endogenous vWF gene in cardiac microvascular endothelial cells (CMEC) was upregulated in the presence of cardiac myocytes. In contrast, endothelial cell levels of thrombomodulin protein and mRNA were unchanged by the addition of ventricular myocytes. Moreover, CMEC expression of vWF was not influenced by the addition of 3T3 fibroblasts or mouse hepatocytes. Taken together, the results suggest that the vWF gene is regulated by vascular bed-specific pathways in response to signals derived from the local microenvironment.

Figure 1

Schematic representation of the vWFlacZ-2 transgene. Arrow, transcriptional start site; SV40 poly(A), SV40 polyadenylation signal; RI, EcoRI; H, HindIII; S, SphI.

Figure 2

The vWFlacZ-2 transgene directs vascular bed–specific expression in vivo. LacZ staining of 10-μm sections from vWFlacZ-2 mouse tissues showing reporter gene activity within the endothelial cell lining of a blood vessel in the white matter of the brain (A), and microvessels of the heart (B) and skeletal muscle (C). In contrast, β-galactosidase activity is not detectable in the lung (D), kidney (E), and spleen (F). The X-Gal reaction product was similarly absent in other organs, including the liver and aorta as well as the megakaryocyte/platelet lineage (data not shown). Bars: (A) 23 μm; (B–F) 63 μm.

Figure 3

The vWFlacZ-2 transgene colocalizes with endogenous vWF within the microvessels of the heart. (A) Whole mounts of the vWFlacZ-2 adult heart incubated with the X-Gal substrate reveals diffuse LacZ staining in both ventricles and atria with distinct sparing of the epicardial coronary arteries (arrowhead). (B) 10-μm section through the left ventricular wall of the vWFlacZ-2 adult heart processed for β-galactosidase activity (blue) and immunoperoxidase detection of vWF (black) reveals co-localization (arrowheads) within the endothelial lining of the microvessels. Bar, 26 μm.

Figure 4

β-galactosidase activity correlates with LacZ mRNA levels. RT-PCR analysis of LacZ, vWF, and TM in vWFlacZ-2 mouse tissues reveals the presence of detectable β-galactosidase transcripts exclusively within the brain, heart, and skeletal muscle of adult transgenic mice. This limited expression pattern contrasts with the more widespread, albeit heterogeneous, distribution of endogenous vWF and TM mRNA in adult mouse tissues. Each lane represents an RT-PCR analysis from identical cDNA template. Two independent experiments in two independent vWFlacZ-2 transgenic lines produced similar results.

Figure 5

Environmental induction of transgene expression in cardiac transplantation model. (A) Whole mount photomicrograph of a 3-wk-old neonatal, wild-type cardiac graft in the ear of an adult vWFlacZ-2 mouse showing the complex network of anastomosing host auricular blood vessels. (B) Two-lead electrocardiogram of a transplanted heart revealing electrocardiographic activity. The heart rate of the graft was 150 beats per min, compared with the native heart rate of 320 beats per min under anesthesia (C) X-Gal staining of a thick 100-μm section from the cardiac graft reveals the presence of β-galactosidase activity in a linear pattern. (D) X-Gal staining of an 8-μm section from the cardiac graft reveals the presence of LacZ-containing endothelial cells next to wild-type ventricular myocytes. (E) X-Gal staining of a 12-μm section through a wild-type lung graft transplanted into the ear of a transgenic mouse that expresses LacZ in all vascular beds. The presence of LacZ-positive blood vessels indicates that the lung graft is revascularized by host-derived endothelium. (F) X-Gal staining of a 12-μm section through a wild-type lung graft in the ear of a vWFlacZ-2 mouse ear revealing absence of detectable LacZ activity. Bars: (C,E,F) 60 μm; (D) 12 μm.

Figure 6

Protein expression in cardiac microvascular endothelial cell-ventricular myocyte coculture. (A) β-Galactosidase activity in CMEC from vWFlacZ-2 mice, as measured with the ONPG substrate, was 2.6-fold higher under coculture conditions (CMEC + myo) compared with either vWFlacZ-2 or wild-type CMEC alone. Antigenic levels of cellular vWF were stimulated 3.1-fold under similar conditions. In contrast, there was no change in the antigenic levels of the endothelial cell marker TM when CMEC was coplated with ventricular myocardial cells. The results are derived from at least three independent experiments, each performed in triplicate. Protein levels are calculated relative to values obtained from primary cultures of vWFlacZ-2 and wild-type–derived CMEC. (B) X-Gal staining of a coculture plate containing CMEC and cardiomyocytes reveals the presence of numerous LacZ-positive endothelial cells integrated within a cluster of myocytes. (C) vWF immunofluorescence under coculture conditions reveals a similar staining pattern with strongly positive endothelial cells interspersed within a colony of cardiomyocytes. Bar, 100 μm.

Figure 6

Protein expression in cardiac microvascular endothelial cell-ventricular myocyte coculture. (A) β-Galactosidase activity in CMEC from vWFlacZ-2 mice, as measured with the ONPG substrate, was 2.6-fold higher under coculture conditions (CMEC + myo) compared with either vWFlacZ-2 or wild-type CMEC alone. Antigenic levels of cellular vWF were stimulated 3.1-fold under similar conditions. In contrast, there was no change in the antigenic levels of the endothelial cell marker TM when CMEC was coplated with ventricular myocardial cells. The results are derived from at least three independent experiments, each performed in triplicate. Protein levels are calculated relative to values obtained from primary cultures of vWFlacZ-2 and wild-type–derived CMEC. (B) X-Gal staining of a coculture plate containing CMEC and cardiomyocytes reveals the presence of numerous LacZ-positive endothelial cells integrated within a cluster of myocytes. (C) vWF immunofluorescence under coculture conditions reveals a similar staining pattern with strongly positive endothelial cells interspersed within a colony of cardiomyocytes. Bar, 100 μm.

Figure 7

Changes in vWF antigen correlate with transcript levels. In ribonuclease protection assays, total RNA from freshly harvested heart (heart), CMEC and CMEC in coculture with ventricular myocytes (CMEC + myo) was hybridized to riboprobes specific for mouse vWF, TM, and β-actin mRNA.