A murine model of myocardial microvascular thrombosis.

Disorders of hemostasis lead to vascular pathology. Endothelium-derived gene products play a critical role in the formation and degradation of fibrin. We sought to characterize the importance of these locally produced factors in the formation of fibrin in the cardiac macrovasculature and microvasculature. This study used mice with modifications of the thrombomodulin (TM) gene, the tissue-type plasminogen activator (tPA) gene, and the urokinase-type plasminogen activator (uPA) gene. The results revealed that tPA played the most important role in local regulation of fibrin deposition in the heart, with lesser contributions by TM and uPA (least significant). Moreover, a synergistic relationship in fibrin formation existed in mice with concomitant modifications of tPA and TM, resulting in myocardial necrosis and depressed cardiac function. The data were fit to a statistical model that may offer a foundation for examination of hemostasis-regulating gene interactions.

PDGF mediates cardiac microvascular communication.

The diversity of cellular and tissue functions within organs requires that local communication circuits control distinct populations of cells. Recently, we reported that cardiac myocytes regulate the expression of both von Willebrand factor (vWF) and a transgene with elements of the vWF promoter in a subpopulation of cardiac microvascular endothelial cells (J. Cell Biol. 138:1117). The present study explores this communication. Histological examination of the cardiac microvasculature revealed colocalization of the vWF transgene with the PDGF alpha-receptor. Transcript analysis demonstrated that in vitro cardiac microvascular endothelial cells constitutively express PDGF-A, but not B. Cardiac myocytes induced endothelial expression of PDGF-B, resulting in PDGF-AB. Protein measurement and transcript analysis revealed that PDGF-AB, but not PDGF-AA, induced endothelial expression of vWF and its transgene. Antibody neutralization of PDGF-AB blocked the myocyte-mediated induction. Immunostaining demonstrated that vWF induction is confined to PDGF alpha-receptor-positive endothelial cells. Similar experiments revealed that the PDGF-AB/alpha-receptor communication also induces expression of vascular endothelial growth factor and Flk-1, critical components of angiogenesis. The existence of this communication pathway was confirmed in vivo. Injection of PDGF-AB neutralizing antibody into the amniotic fluid surrounding murine embryos extinguished expression of the transgene. In summary, these studies suggest that environmental induction of PDGF-AB/alpha-receptor interaction is central to the regulation of cardiac microvascular endothelial cell hemostatic and angiogenic activity.

Mutations in PAX3 associated with Waardenburg syndrome type I.

Waardenburg syndrome (WS) types I, II, and III (McKusick #14882, #19351, and #19350) are related autosomal dominant disorders characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Disease causing PAX3 mutations have been identified in a few families from each of the three disease subtypes, WS-I, WS-II, and WS-III. In others, although the mutations have not been pinpointed, linkage with the PAX3 locus on chromosome 2q35 has been demonstrated. The PAX3 protein is a transcription factor that contains both a paired-domain and a homeodomain DNA binding motif and appears to play a key role during embryogenesis. In this report, we describe two mutations in the human PAX3 gene that cause WS type I. One mutation is a deletion/frameshift in the paired-domain of PAX3 and results in a protein without functional DNA binding domains. The second mutation is a single-base substitution and results in a premature termination codon in the homeodomain of PAX3. This is the first demonstration of a mutation in the homeodomain DNA binding motif in this protein resulting in WS and one of the few examples of a mutation in a homeodomain of any protein that results in human disease.

Age-related changes in fibronectin expression in spontaneously hypertensive, Wistar-Kyoto, and Wistar rat hearts.

The effects of age and blood pressure on fibronectin expression in the rat heart were studied in the normotensive Wistar and Wistar-Kyoto (WKY) strains and in the spontaneously hypertensive rat (SHR). Fibronectin mRNA expression decreased threefold between 10 and 40 weeks of age in Wistar hearts, with changes of similar magnitude occurring between 6 and 24 weeks in WKY rats. In contrast, no decrease in fibronectin mRNA was observed in SHR hearts during this time span. These results are in contrast to changes observed previously in the aorta, where an increase in fibronectin mRNA occurred with age in all three rat strains. Ribonuclease protection analysis showed a small age-specific increase in the relative content of EIIIA+ fibronectin mRNA isoforms in hearts from Wistar rats, whereas no change was found in the relative amount of either EIIIA or EIIIB isoforms in SHR hearts. Changes similar to those observed for fibronectin mRNA, although of different magnitudes, were observed in mRNA levels for collagen alpha 1(III) and beta 1 integrin. In Wistar hearts, collagen alpha 1(III) mRNA levels decreased fivefold to sixfold between 10 and 40 weeks of age, whereas a twofold to threefold decrease in beta 1 integrin was observed in WKY hearts between 6 and 24 weeks of age. Western blot analysis revealed a positive correlation between fibronectin mRNA and protein for age-dependent changes in ventricular tissue but not in the atria, suggesting that the regulation of fibronectin expression during the changes common to both aging and hypertrophy could involve both transcriptional and posttranscriptional mechanisms.

Hypertension induces alternatively spliced forms of fibronectin in rat aorta.

Fibronectin expression was shown recently to increase in the rat aorta in response to experimental hypertension. Fibronectin is known to alter the phenotype of vascular smooth muscle and endothelial cells, and relative changes in the expression of different isoforms of fibronectin, generated by alternative splicing and distinguished by the absence or presence of inserts designated as EIIIA, EIIIB, and V, may reflect a change in cell phenotype. In the present study we examined the expression of alternatively spliced forms of aortic fibronectin during deoxycorticosterone-salt hypertension. Aortic RNA was analyzed quantitatively using Northern blot analysis and ribonuclease protection assays. Using Northern blot analysis, deoxycorticosterone-salt treatment for 21 days led to a 4.9-fold increase in EIIIA fibronectin messenger RNA, while EIIIB and V forms increased by 2.6- and 2.5-fold, respectively. As determined by ribonuclease protection assays, the percentage of fibronectin transcripts containing either EIIIA, EIIIB, or V in control aorta was 7.3%, 19%, and 40%, respectively. The percentage of EIIIA transcripts increased 42% over control levels after 21 days of deoxycorticosterone-salt treatment, whereas no proportionate change in the other alternatively spliced forms was found. Thus, all forms increased, but a selective increase in the EIIIA form was induced. Analogous increases in each of the fibronectin isoforms were found in the spontaneously hypertensive rats when compared with age-matched Wistar-Kyoto or Wistar rats, and 40-week-old animals showed increases over 10-week-old animals in all strains, consistent with an age-dependent increase in aortic fibronectin expression.

Rapid expression of fibronectin in the rabbit heart after myocardial infarction with and without reperfusion.

The expression of fibronectin in the repair process after myocardial infarction was studied using two protocols of coronary occlusion in the rabbit: a permanent occlusion or 3 h of occlusion followed by reperfusion (too late for salvage). We found a rapid and progressive increase in cardiac fibronectin expression in the infarcted region of the ventricle. Steady-state mRNA levels for fibronectin increased 13- and 16-fold, respectively, in the permanent and reperfused infarcts 1 d postinfarction. Immunological detection of the protein with a polyclonal antibody against plasma fibronectin showed significant increases of the protein fibronectin in the infarcted myocardium by day 3 in the reperfused group and by day 5 in the permanent coronary occlusion group. Ribonuclease protection assays established the induction of EIIIB containing fibronectin mRNA in both models by day 1 and use of a monoclonal antibody showed an increase in the EIIIA isoform 2 d postinfarction. Increases in steady-state mRNA levels for several collagen types were found in both groups, but these changes occurred after those noted for fibronectin. Thus fibronectin mRNA and protein expression increased rapidly postinfarction suggesting a functional role in the repair process.

Fibronectin expression in the normal and hypertrophic rat heart.

We examined changes in the expression of fibronectin during the induction of cardiac hypertrophy by L-triiodothyronine administration and by mineralocorticoid- and salt-induced experimental hypertension. By use of Northern and Western blotting procedures, fibronectin was localized mainly in the atria of normal rat hearts. Atria contained 10- and 5-fold higher relative concentrations of fibronectin mRNA and protein, respectively, compared with ventricles. During the progression of cardiac hypertrophy induced by L-triiodothyronine over a 10-d period, there was a progressive increase in fibronectin mRNA for the first 6 d followed by a return to control levels. The major change could be accounted for by changes in ventricular mRNA, which increased about four- to sixfold. In contrast, protein levels in ventricles increased progressively over the 10-d treatment period. Ribonuclease protection analysis indicated that the relative amounts of fibronectin isoforms containing exons designated EIIIA and EIIIB increased during the progression of hypertrophy. When cardiac hypertrophy was induced by mineralocorticoid and salt treatment, increases in ventricular fibronectin mRNA and protein and the induction of alternatively spliced forms of fibronectin were also observed. However, the extent and temporal pattern of fibronectin expression differed between the two experimental models.