Moderation of Calpain Activity Promotes Neovascular Integration and Lumen Formation during VEGF-Induced Pathological Angiogenesis

Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF

Rho activity critically and selectively regulates endothelial cell organization during angiogenesis

The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants. In this animal model, dominant negative N19RhoA selectively impaired assembly of ECs into new blood vessels; and, in contrast, active V14RhoA stimulated ECs to form blood vessels with functional lumens. In vitro, dominant negative N19RhoA reduced EC actin stress fibers and prevented ECs from contracting and reorganizing into precapillary cords within collagen gels. In contrast, active V14RhoA promoted EC stress fiber formation, contractility, and organization into cords. Neither N19RhoA nor V14RhoA significantly affected EC proliferation or migration in vitro; and, similarly, neither mutant significantly affected EC density during angiogenesis in vivo. Thus, these studies identify a critical and selective role for Rho activity in regulating EC assembly into new blood vessels, and they identify both negative and positive manipulation of Rho activity, respectively, as strategies for suppressing or promoting the organizational stages of neovascularization

Activation of the Nrf2 Cell Defense Pathway by Ancient Foods: Disease Prevention by Important Molecules and Microbes Lost from the Modern Western Diet

The Nrf2 (NFE2L2) cell defense pathway protects against oxidative stress and disorders including cancer and neurodegeneration. Although activated modestly by oxidative stress alone, robust activation of the Nrf2 defense mechanism requires the additional presence of co-factors that facilitate electron exchange. Various molecules exhibit this co-factor function, including sulforaphane from cruciferous vegetables. However, natural co-factors that are potent and widely available from dietary sources have not been identified previously. The objectives of this study were to investigate support of the Nrf2 cell defense pathway by the alkyl catechols: 4-methylcatechol, 4-vinylcatechol, and 4-ethylcatechol. These small electrochemicals are naturally available from numerous sources but have not received attention. Findings reported here illustrate that these compounds are indeed potent co-factors for activation of the Nrf2 pathway both in vitro and in vivo. Each strongly supports expression of Nrf2 target genes in a variety of human cell types; and, in addition, 4-ethylcatechol is orally active in mice. Furthermore, findings reported here identify important and previously unrecognized sources of these compounds, arising from biotransformation of common plant compounds by lactobacilli that express phenolic acid decarboxylase. Thus, for example, Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus collinoides, which are consumed from a diet rich in traditionally fermented foods and beverages, convert common phenolic acids found in fruits and vegetables to 4-vinylcatechol and/or 4-ethylcatechol. In addition, all of the alkyl catechols are found in wood smoke that was used widely for food preservation. Thus, the potentially numerous sources of alkyl catechols in traditional foods suggest that these co-factors were common in ancient diets. However, with radical changes in food preservation, alkyl catechols have been lost from modern foods. The absence of alkyl catechols from the modern Western diet suggests serious negative consequences for Nrf2 cell defense, resulting in reduced protection against multiple chronic diseases associated with oxidative stress

Model for bioconversion of inactive dietary precursors to Nrf2 activators by phenolic acid decarboxylase (PAD).

<p>The microbial enzyme, PAD, expressed by <i>Lactobacillus plantarum</i>, <i>Lactobacillus brevis</i>, and other, but not all, lactobacillus strains convert caffeic acid (inactive) to 4-vinylcatechol (Nrf2 activator). Similarly, PAD converts 3,4-dihydroxybenzoic acid (inactive) to catechol (Nrf2 activator). See text for references and subsequent figures for supporting data.</p

Biotransformation of caffeic acid and 3,4-dihydroxybenzoic acid, as demonstrated with western blotting.

<p>(A) Biotransformation by <i>L</i>. <i>plantarum</i> as demonstrated with western blotting of human dermal microvascular endothelial cells, harvested 24 hours after addition of test samples. Blots were stained for the Nrf2 target gene HO-1, Nrf2 itself, and CD31 as loading control. Key: Ctrl = control, CFA = caffeic acid, LP = control supernatant from <i>L</i>. <i>plantarum</i> incubated with PBS-glucose and filter-sterilized, (LP + CFA) = supernatant from <i>L</i>. <i>plantarum</i> incubated with CFA in PBS-glucose and filter-sterilized, 3,4-DHBA = 3,4-dihydroxybenzoic acid, (LP + 3,4-DHBA) = supernatant from <i>L</i>. <i>plantarum</i> + 3,4-DHBA incubated in PBS-glucose and filter-sterilized. CFA, 3,4-DHBA, and lactobacillus-incubations with each were added to a final concentration corresponding to 30 μM CFA and 30 μM 3,4-DHBA starting material (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#sec002" target="_blank">Methods</a>). Positive controls = catechol (30 μM) and 4-ethylcatechol (4EC, 30 μM). (B) Biotransformation by <i>L</i>. <i>brevis</i> (LB), with experimental conditions otherwise identical to panel (A), above. Also, for experiment shown in panel (B), 4EC = 4-ethylcatechol positive control was added to a final concentration of 15 μM instead of 30 μM.</p