Severe deficiency of coagulation Factor VII results in spontaneous cardiac fibrosis in mice.

Mice genetically modified to produce low levels (approximately 1% of wild-type) of coagulation FVII presented with echocardiographic evidence of heart abnormalities. Decreases in ventricular size and reductions in systolic and diastolic functions were found, suggestive of a restrictive cardiomyopathy and consistent with an infiltrative myopathic process. Microscopic analysis of mouse hearts showed severe patchy fibrosis in the low-FVII mice. Haemosiderin deposition was discovered in hearts of these mice, along with increases in inflammatory cell number, ultimately resulting in widespread collagen deposition. Significant increases in mRNA levels of TGFbeta, TNFalpha and several matrix metalloproteinases in low-FVII mice, beginning at early ages, supported a state of cardiac remodelling associated with the fibrotic pathology. Mechanistic time-course studies suggested that cardiac fibrosis in low-FVII mice originated from bleeding in heart tissue, resulting in the recruitment of leukocytes, which released inflammatory mediators and induced collagen synthesis and secretion. These events led to necrosis of cardiomyocytes and collagen deposition, characteristics of cardiac fibrosis. The results of this study demonstrated that haemorrhagic and inflammatory responses to a severe FVII deficiency resulted in the development of cardiac fibrosis, observed echocardiographically as a restrictive cardiomyopathy, with compromised ventricular diastolic and systolic functions.

Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi.

A minimally invasive laser-induced injury model is described to study thrombus development in mice in vivo. The protocol involves focusing the beam of an argon-ion laser through a compound microscope on the vasculature of a mouse ear that is sufficiently thin such that blood flow can be visualized by intravital microscopy. Two distinct injury models have been established. The first involves direct laser illumination with a short, high-intensity pulse. In this case, thrombus formation is inhibited by the GPIIb/IIIa antagonist, G4120. However, the anticoagulants, hirulog, PPACK, and NapC2 have minimal effect. This indicates that thrombus development induced by this model mainly involves platelet interactions. The second model involves low-intensity laser illumination of mice injected with Rose Bengal dye to induce photochemical injury in the region of laser illumination. Thrombi generated by this latter procedure have a slower development and are inhibited by both anticoagulant and anti-platelet compounds.

The characterization of mice with a targeted combined deficiency of protein c and factor XI.

Activated protein C functions directly as an anticoagulant and indirectly as a profibrinolytic enzyme. To determine whether the fibrin deposition previously observed in PC(-/-) murine embryos and neonates was mediated through the FXI pathway, PC(+/-)/FXI(-/-) mice were generated and crossbred to produce double-deficient progeny (PC(-/-)/FXI(-/-)). PC(-/-)/FXI(-/-) mice survived the early lethality observed in the PC(-/-)/FXI(+/+) neonates, with the oldest PC(-/-)/FXI(-/-) animal living to 3 months of age. However, the majority of these animals was sedentary and significantly growth-retarded. On sacrifice or natural death, all of these PC(-/-)/FXI(-/-) mice demonstrated massive systemic fibrin deposition with concomitant hemorrhage and fibrosis, as confirmed through histological analyses. Several of these animals also presented with enlarged lymph nodes and extensive lymphatic fluid in the thoracic cavity. Thus, although a number of the PC(-/-)/FXI(-/-) mice survived the lethal perinatal coagulopathy seen in the PC(-/-) neonates, they nonetheless succumbed to overwhelming thrombotic disease in later life. This combined deficiency state provided the first clear indication that the course of a severe thrombotic disorder could be manipulated by blocking the intrinsic pathway and provided the first opportunity to study a total protein C deficiency in an adult animal.

Remodeling of the vessel wall after copper-induced injury is highly attenuated in mice with a total deficiency of plasminogen activator inhibitor-1.

Clinical studies have indicated that high plasma levels of fibrinogen, or decreased fibrinolytic potential, are conducive to an increased risk of cardiovascular disease. Other investigations have shown that insoluble fibrin promotes atherosclerotic lesion formation by affecting smooth muscle cell proliferation, collagen deposition, and cholesterol accumulation. To directly assess the physiological impact of an imbalanced fibrinolytic system on both early and late stages of this disease, mice deficient for plasminogen activator inhibitor-1 (PAI-1(-/-)) were used in a model of vascular injury/repair, and the resulting phenotype compared to that of wild-type (WT) mice. A copper-induced arterial injury was found to generate a lesion with characteristics similar to many of the clinical features of atherosclerosis. Fibrin deposition in the injured arterial wall at early (7 days) and late (21 days) times after copper cuff placement was prevalent in WT mice, but was greatly diminished in PAI-1(-/-) mice. A multilayered neointima with enhanced collagen deposition was evident at day 21 in WT mice. In contrast, only diffuse fibrin was identified in the adventitial compartments of arteries from PAI-1(-/-) mice, with no evidence of a neointima. Neovascularization was observed in the adventitia and was more extensive in WT arteries, relative to PAI-1(-/-) arteries. Additionally, enhanced PAI-1 expression and fat deposition were seen only in the arterial walls of WT mice. The results of this study emphasize the involvement of the fibrinolytic system in vascular repair processes after injury and indicate that alterations in the fibrinolytic balance in the vessel wall have a profound effect on the development and progression of vascular lesion formation.

Fluid secretion in Rhodnius upper malpighian tubules (UMT): water osmotic permeabilities and morphometric studies.

We have measured the osmotic permeability of the basolateral cell membrane (Poscb) and compared it with the transepithelial permeability (Poste) to calculate the paracellular (Posp) permeability of the upper malpighian tubules (UMT) of the 5th instar of Rhodnius prolixus under several experimental conditions, namely, at rest and after stimulation to secrete with 5-HT, each under control conditions (no treatment), after treatment with pCMBS, and after addition of pCMBS and DTT. Secretion rate is negligible at rest. During stimulation mean secretion rate is 43.5 nl/cm2 sec. Secretion is severely curtailed by pCMBS and fully restored by DTT. Poscb = 9.4 (resting, control); 5.8 (control + pCMBS); 10.7 (control + pCMBS + DTT); 20.6 (stimulated, control); 14.7 (stimulated + pCMBS); 49.1 (stimulated + pCMBS + DTT) (x10?4 cm3/cm2 sec Osm). Calculated Posp are higher than the transcellular permeability, Posc, at rest and after stimulation. Electron micrograph morphometry of UMT sections show that cells significantly decrease their volume after stimulation. Lateral intercellular space (LIS) and basolateral extracellular labyrinth (BEL) are barely discernible at rest. LIS and BEL are widely dilated in stimulated UMT. Thus, ions have restricted access to the deep and narrow basolateral cell membrane indentations at rest, but they have ready access to cell membrane indentations after stimulation, because of the opening of LIS and BEL. These findings are discussed in relation to isosmotic secretion. The rate-limiting step for paracellular movement is located at the smooth septate junctions.

Tumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1.

In vivo tumor progression in mice with targeted deficiencies in urokinase-type plasminogen activator (UPA-/-) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1-/-), was studied using a fibrosarcoma tumor model. Murine T241 fibrosarcoma cells were s.c. implanted into three groups of mice with the following genotypes, wild-type (WT), UPA-/-, and PAI-1-/-. A significantly diminished primary tumor growth in UPA-/- and PAI-1-/- mice occurred, relative to WT mice. Tumors in UPA-/- and PAI-1-/- mice displayed lower proliferative and higher apoptotic indices and displayed a different neovascular morphology, as compared with WT mice. These results are consistent with the decreased growth rates of this tumor in these gene-deleted mice. Immunohistochemical analyses of the tumors revealed a decrease in vascularity and vascular endothelial growth factor expression only in tumors in PAI-1-/- mice. Analyses of the relative extents of corneal angiogenesis in these same animals, induced by basic fibroblast growth factor, corroborated the resistance of PAI-1-/- mice to neovascularization. The results obtained suggest that the host fibrinolytic system plays an important role in tumor growth in this model. Alterations in host expression of components of this system may alter tumor growth and dissemination by affecting the balance between tumor cell death and proliferation, as well as extracellular matrix changes needed for invasiveness and angiogenesis.