Foxc1 is required by pericytes during fetal brain angiogenesis

Summary Brain pericytes play a critical role in blood vessel stability and blood–brain barrier maturation. Despite this, how brain pericytes function in these different capacities is only beginning to be understood. Here we show that the forkhead transcription factor Foxc1 is expressed by brain pericytes during development and is critical for pericyte regulation of vascular development in the fetal brain. Conditional deletion of Foxc1 from pericytes and vascular smooth muscle cells leads to late-gestation cerebral micro-hemorrhages as well as pericyte and endothelial cell hyperplasia due to increased proliferation of both cell types. Conditional Foxc1 mutants do not have widespread defects in BBB maturation, though focal breakdown of BBB integrity is observed in large, dysplastic vessels. qPCR profiling of brain microvessels isolated from conditional mutants showed alterations in pericyte-expressed proteoglycans while other genes previously implicated in pericyte–endothelial cell interactions were unchanged. Collectively these data point towards an important role for Foxc1 in certain brain pericyte functions (e.g. vessel morphogenesis) but not others (e.g. barriergenesis)

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

Immunohistochemical staining of Nrf2 in human endothelial cells.

<p>Cells were incubated with compounds (30 μM final concentration) for 24 hours, fixed, and stained for Nrf2 (green color) and F-actin (red color). Vehicle ctrl = vehicle control, 4EP = 4-ethylphenol, (negative control), 2M4M = 2-methoxy-4-methylphenol (negative control), CFA = caffeic acid (negative control), 4MC = 4-methylcatechol, 4VC = 4-vinylcatechol, 4EC = 4-ethylcatechol. Note bright green staining of Nrf2 in cells stimulated with catechol, 4MC, 4VC, and 4EC, in comparison with controls. See Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g005" target="_blank">5</a> for all chemical structures. All samples were processed and stained in parallel; green images (Nrf2) were captured at identical exposure; and, similarly, red images (F-actin) were captured at identical exposure. Subsequently, red and green images were merged without any manipulation so that images presented here are valid for direct comparisons.</p

Model for multi-step bioconversion of inactive dietary precursors to Nrf2 activators by <i>Lactobacillus collinoides</i>.

<p>Microbial cinnamoyl esterase from <i>L</i>. <i>collinoides</i> converts chlorogenic acid (inactive) to caffeic acid (inactive), thereby providing substrate for phenolic acid decarboxylase (PAD)-mediated generation of 4-vinyl catechol (Nrf2 activator). Finally, microbial phenolic acid reductase, also expressed by <i>L</i>. <i>collinoides</i>, reduces 4-vinylcatechol to 4-ethylcatechol (Nrf2 activator). See text for supporting references and subsequent figures for supporting data.</p

Activation of the Nrf2 pathway by alkyl catechols and catechol is regulated by oxygen.

<p>Human microvascular endothelial cells were cultured in 21% oxygen (room air) or 2% oxygen (hypoxia), as indicated, and stimulated with the specified compounds (20 μM each) for 24 hours. RT-PCR, as above, was used to quantify mRNAs; Y-axis = (mRNA copies)/(10⁶ 18S rRNA copies). Cat = catechol, 4MC = 4-methylcatechol, 4EC = 4-ethylcatechol, HQ = hydroquinone, TBHQ = tert-butylhydroquinone (HQ and TBHQ are well known activators of Nrf2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g001" target="_blank">Fig 1</a> for chemical structures). Nrf2 target genes = HO-1, NQO1, G6PD; CD31 (PECAM-1) = internal control; GLUT1 (glucose transporter 1) is induced by hypoxia and serves as a positive control for hypoxia-induced gene expression. Error bars = ± S.D.; n ≥ 3 for each data point. <b><i>Summary of data analyses and statistical significance</i>:</b> Again, it is important to emphasize that the Nrf2 pathway is activated primarily by stabilization of Nrf2 protein that allows for transcriptional induction of Nrf2 target genes, such as HO-1, NQO1, and G6PD; and therefore, these target gene mRNAs are indicators of Nrf2 pathway activation. Activation of the Nrf2 pathway is not mediated primarily by induction of Nrf2 mRNA, but Nrf2 mRNA induction may contribute modestly as suggested by data shown here in the Nrf2 data panel. Thus, for the HO-1 and NQO1 data sets, representing activation of the Nrf2 pathway, individual comparisons between a specific compound (<i>i</i>.<i>e</i>. Cat, 4MC, 4EC, HQ, or TBHQ), used at 21% oxygen vs. 2% oxygen, indicated oxygen-dependent differences that are all extremely significant (p<0.001). For the G6PD data set, also representing activation of the Nrf2 pathway, individual comparisons between a specific compound used at 21% oxygen vs. 2% oxygen indicated oxygen-dependent differences that are all very significant (p<0.01) with the exception of Cat (p<0.05, significant). Also, for HO-1 and NQO1, additional comparisons for each of the compounds vs. corresponding Ctrl = extremely significant (p<0.0001) for both 21% oxygen and 2% oxygen. For the G6PD data panel and for both 21% oxygen and 2% oxygen: Ctrl vs. Cat (p<0.005, very significant); Ctrl vs. 4MC, Ctrl vs. 4EC, Ctrl vs. HQ, Ctrl vs. TBHQ (all p<0.0002, extremely significant). For Nrf2, and for both 21% oxygen and 2% oxygen data sets: Ctrl vs. each of the compounds = significant (p≤0.03), with the exception of Cat (21% oxygen) = not significant. For CD31, and for both 21% oxygen and 2% oxygen data sets: Ctrl vs. each of the compounds = not significant, with the exception of Ctrl vs. 4EC and Ctrl vs. TBHQ (21% oxygen) = very significant (p<0.01). Nonetheless, these differences are relatively small in comparison with the large inductions of Nrf2 target gene expression shown in HO-1 and NQO1 data panels. Finally, for the 2% oxygen GLUT1 panel, representing induction of GLUT1 by hypoxia, individual comparisons between Ctrl and each of the compounds indicated differences that were all very significant (p<0.01 to p<0.001). Also, individual comparisons between each experimental group in 2% oxygen with the corresponding experimental group in 21% oxygen indicated differences that were all extremely significant (p<0.0001).</p

Biotransformation of caffeic acid by <i>L</i>. <i>collinoides</i>, as demonstrated with HPLC.

<p>Y-axis = absorbance at 254nm (mAU), X-axis = minutes. <u>Top panel:</u> HPLC of caffeic acid standard. <u>Middle panel:</u> HPLC of 4-vinylcatechol and 4-ethylcatechol standards. <u>Bottom panel:</u> HPLC of supernatant from caffeic acid + <i>L</i>. <i>collinoides</i> incubation, consistent with bioconversion of caffeic acid to 4-ethylcatechol. Retention times: caffeic acid = 8.1 minutes, 4-vinylcatechol = 10.7 minutes, 4-ethylcatchol = 11.6 minutes.</p

Compounds with structural similarity to catechols that do not activate the Nrf2 pathway significantly, in comparison with catechol or akyl catechols.

<p>All compounds depicted here were tested in RT-PCR assays and/or western blotting assays with human endothelial cells, as demonstrated in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g003" target="_blank">3</a> at a final concentration of 30 μM, with the exception of quercetin and luteolin that were tested at 20 μM (the maximum tolerated dose). None of these compounds induced Nrf2 target gene expression significantly in comparison with catechol or the akyl catechols. Consistent with these negative findings, each of these compounds has structural characteristics consistent with inactivity, either due to methylation of hydroxyls (top panel), lack of appropriate hydroxyls on benzene ring (middle panel), or electron-withdrawing or bulky side groups appended to the catechol moiety (bottom panel). For supporting data, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.g003" target="_blank">Fig 3A</a> (for 4-ethylphenol) and subsequent figures (for caffeic acid, chlorogenic acid, and 3,4-dihydroxybenzoic acid) and also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#pone.0148042.s003" target="_blank">S3 Fig</a> (for all other compounds).</p

Biotransformation of chlorogenic acid and caffeic acid by <i>Lactobacillus collinoides</i>, as measured with RT-PCR.

<p>Y-axis = (mRNA copies)/(10⁶ 18S rRNA copies). RNA was isolated from human dermal microvascular endothelial cells, 24 hours after addition of test samples: Ctrl = control, CA = chlorogenic acid, CFA = caffeic acid, LC = control supernatant from <i>L</i>. <i>collinoides</i> incubated with PBS-glucose and filter-sterilized, (LC + CA) = supernatant from <i>L</i>. <i>collinoides</i> incubated with CA in PBS-glucose and filter-sterilized, (LC + CFA) = supernatant from <i>L</i>. <i>collinoides</i> incubated with CFA in PBS-glucose and filter-sterilized. CA, CFA, and <i>L</i>. <i>collinoides</i> incubations with each were added to a final concentration corresponding to 30 μM CA and 30 μM CFA starting material (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148042#sec002" target="_blank">Methods</a>). (4EC) = 4-ethylcatechol positive control (30 μM). Nrf2 target genes = HO-1, NQO1, G6PD; control mRNAs = CD31 and VE-cadherin. Error bars = ± S.D.; n ≥ 3 for each data point. <b><i>Summary of data analyses and statistical significance</i>:</b> As discussed previously, the Nrf2 pathway is activated primarily by stabilization of Nrf2 protein that allows for transcriptional induction of Nrf2 target genes, such as HO-1, NQO1, and G6PD; and therefore, these target gene mRNAs are indicators of Nrf2 pathway activation. Activation of the Nrf2 pathway is not mediated primarily by induction of Nrf2 mRNA, but Nrf2 mRNA induction may contribute modestly as suggested by data shown here (see text for further explanation and references). Thus, for HO-1, NQO1 and G6PD data panels, individual comparisons for Ctrl vs. LC+CA, Ctrl vs. LC+CFA, and Ctrl vs. 4EC indicated differences that are all extremely statistically significant (p< 0.0001). In contrast, individual comparisons for Ctrl vs. CA, Ctrl vs. CFA, and Ctrl vs. LC indicated no significant differences. For Nrf2, Ctrl vs. CA, Ctrl vs. CFA, and Ctr vs. LC = no significant differences; Ctrl vs. LC+CA, Ctrl vs. LC+CFA, and Ctrl vs. 4EC = all very significant differences (p<0.01). For CD31 and VE-cadherin: no statistically significant differences.</p

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