Vascular permeability is an important indicator for the physiological status of blood vessels. Increases in microvascular permeability have been demonstrated in a number of systemic diseases (e.g., diabetes, hypertension, and rheumatoid arthritis) (37
). Inflammatory mediators (e.g., histamine, serotonin, and bradykinin), VEGF, and shear stress have been shown to increase vascular permeability (38
). The mechanisms leading to the increased permeability are thought to occur either by passing through openings between adjacent ECs (intercellular) or by passing through the peripheral cytoplasm of ECs (transcellular) (37
). We observed increased vascular leakage of Evans blue dye in BMK1-CKO mice as early as 1 week after induction of Cre recombinase when no hemorrhages were found in any organs. The increased leakage after BMK1 ablation is probably partially due to the increased permeability of ECs through transcellular openings, since numerous vesicles and vacuoles were found in a number of ECs (Figure C). It is still possible, however, that some of the leakage might pass through intercellular openings because complicated arrangements of fingerlike processes that interleave with each other were also found in the ultrastructural analysis of the capillaries.
The survival of ECs is vital for the maintenance of vasculature integrity. Growth factors such as VEGF, bFGF, and angiopoietin-1 are recognized to sustain EC survival by preventing EC apoptosis (40
). The inhibition of EC apoptosis by these growth factors is shown to be dependent on the activation of intracellular PI3K/Akt signaling pathway (31
) and may also be dependent on the upregulation of antiapoptotic proteins such as survivin and Bcl-2 (43
). Herein, we demonstrated that in addition to PI3K/Akt, the BMK1 pathway is also required for EC survival, possibly through transmitting essential antiapoptotic signals from extracellular agonists. Besides the survival signals from endothelial growth factors, however, EC matrix and/or EC-EC contacts also have been shown to support cell survival (46
). Therefore, the role of the BMK1 pathway in integrands or EC homophilic adhesion-mediated antiapoptotic signaling needs to be elucidated.
Other than growth factors, oxidative stress is also a major activator for the BMK1 pathway (48
). Reactive oxygen species are known to play a critical role in inducing apoptosis, and highly reactive oxygen species levels have been detected in a number of human diseases such as aging, ischemia, cancer, atherosclerosis, and neurodegenerative disease (50
). The activation of BMK1 has been demonstrated to provide the survival signal for neutralizing cellular damage caused by oxidative insults (35
). It is of interest to investigate whether the BMK1 pathway also provides survival signal for ECs to prevent the pathogenesis of human disorders caused by oxidative injury.
The mechanism linking the apoptosis of ECs to the formation of lumplike masses in heart tissues is unknown (Figure A). These masses, however, are clearly not generated by the defects of cardiomyocytes lacking BMK1, since the efficiency of Cre-mediated recombination in these cells is extremely low. Moreover, data from BMK1-cmKO mice strongly support the notion that BMK1 is not required for cardiac development and baseline function. One of the explanations is that the heart may be a unique tissue in which minor, repetitive mechanical injury to the already weakened ECs of BMK1-CKO mice produces repeated hemorrhaging, which, in combination with the consequential inflammation, leads to the generation of these masses. In addition, other genetic and physiological factors such as the embryologic origin of cardiac ECs, blood flow variation in different vessel types, as well as cardiac tissue oxygenation level, may also contribute to these differential phenotypes.
Any given MAPK contributes to the specificity of cellular responses, in part, through its particular downstream substrates. To date, many downstream targets of BMK1 have been identified, and, among them, the members of the MEF2 family of transcriptional factors are the best-characterized targets (1
). This family of transcriptional factors is composed of four members that were initially found as muscle-specific DNA-binding proteins, which recognized MEF2 motifs located inside the promoters of a number of muscle-specific genes (56
). Phosphorylation of MEF2 by BMK1 activates MEF2. This activation has been shown to be critical for growth factor–induced neuronal survival (8
). In mice, MEF2C is expressed in developing cardiomyocytes, ECs, and smooth muscle cells, as well as in the surrounding mesenchyme, during embryogenesis. Disruption of the MEF2C
locus leads to cardiovascular defects (14
) bearing striking similarity to the embryonic abnormality observed in the BMK1-deficient mutant. The fact that activated MEF2C can partially rescue EC apoptosis caused by BMK1 removal further supports the concept that the BMK1 pathway relays its antiapoptotic signal within ECs through activating its downstream target MEF2C. The partial rescue of EC apoptosis suggests, however, that other mechanisms might also be involved, such as BMK1-dependent phosphorylation of Bcl2 antagonist of cell death (Bad), which was most recently described by Berk’s lab (57
The malfunction of both ECs and myocardiac cells has been suspected to play a part for the cardiovascular defects observed in BMK1-KO mutants (11
). These complex phenotypes of the conventional BMK1-KO mutant lead to fatalities in embryos, making it difficult to distinguish them from primary to secondary phenomena. We have circumvented this dilemma by generating mice with BMK1 ablation specifically in endothelium or in cardiac myocytes. EC-specific BMK1-KO mutant reproduced the cardiovascular defect of BMK1-KO mice, while cardiomyocyte-specific BMK1 KO mice developed normally and survived for more than 1 year without any cardiac complication. These results strongly suggest that BMK1 is essential for the function of ECs and the cardiac defects observed in conventional BMK1-KO mice is a secondary event resulting from dysfunctional ECs. Although BMK1-cmKO mice appear to be normal, suggesting that the BMK1 pathway is not critical for development and general maintenance of cardiomyocytes, a number of studies have shown that the BMK1 pathway plays a role in various pathogenic processes of cardiac disease involving cardiomyocyte (58
). Currently, we are evaluating the function of BMK1 in cardiomyocytes under stressed conditions using BMK1-cmKO mice.
In conclusion, we have shown that conditional ablation of BMK1 in adult mice results in defects of vascular integrity and endothelial maintenance. Moreover, we have also demonstrated in vitro that an intact signaling pathway in BMK1 is essential for EC survival. The phenotype observed here is of particular interest since, thus far, no genetic model with “endothelial failure” in the adult stage has been demonstrated. Moreover, the role for endothelial apoptosis as a mechanism for the effect of BMK1 inhibition in ECs may facilitate the design of effective therapy to enhance angiogenesis in diseases of tissue ischemia or inhibit angiogenesis in diseases dependent on neovascularization.