Plasminogen activator inhibitor-1 regulates myoendothelial junction formation

Abstract

Rationale: Plasminogen activator inhibitor-1 (PAI-1) is a biomarker for several vascular disease states; however, its target of action within the vessel wall is undefined.

Objective: Determine the ability of PAI-1 to regulate myoendothelial junction (MEJ) formation.

Methods and results: MEJs are found throughout the vasculature linking endothelial cells (ECs) and vascular smooth muscle cells. Using a vascular cell coculture we isolated MEJ fractions and performed two-dimensional differential gel electrophoresis. Mass spectrometry identified PAI-1 as being enriched within MEJ fractions, which we confirmed in vivo. In the vascular cell coculture, recombinant PAI-1 added to the EC monolayer significantly increased MEJs. Conversely, addition of a PAI-1 monoclonal antibody to the EC monolayer reduced the number of MEJs. This was also observed in vivo where mice fed a high fat diet had increased PAI-1 and MEJs and the number of MEJs in coronary arterioles of PAI-1(-/-) mice was significantly reduced when compared to C57Bl/6 mice. The presence of MEJs in PAI-1(-/-) coronary arterioles was restored when their hearts were transplanted into and exposed to the circulation of C57Bl/6 mice. Application of biotin-conjugated PAI-1 to the EC monolayer in vitro confirmed the ability of luminal PAI-1 to translocate to the MEJ. Functionally, phenylephrine-induced heterocellular calcium communication in the vascular cell coculture was temporally enhanced when recombinant PAI-1 was present, and prolonged when PAI-1 was absent.

Conclusion: Our data implicate circulating PAI-1 as a key regulator of MEJ formation and a potential target for pharmacological intervention in diseases with vascular abnormalities (eg, diabetes mellitus).

Figure 1. Isolation of MEJ protein fractions from vascular cell co-culture

Confocal microscopy of VCCC stained with phalloidin en face (A–C) and transverse (D–F). Conditions shown include unscraped membranes viewed en face (A) and transverse (D) scraped membranes viewed enface (B) and transverse (E) as well as scraped and lysed membranes viewed en face (C) and transverse (F). SDS-PAGE of VSMC, EC and MEJ fractions stained with GelCode Blue (G) and silver stain (H). Immunoblots of protein fractions probed for VE-Cadherin (I), SMα-actin (J) and GAPDH (K). Bar in A is 20 µm and representative for A–C; bar in D is 10 µm and representative for D–F. Arrows in A and B indicate pores of the Transwell insert.

Figure 2. 2D-DIGE analysis of isolated MEJ protein fractions from vascular cell co-culture

2D-DIGE blots of isolated protein fractions from the VCCC. Cartoon schematics in A represent the compared fractions for each gel image as they occur in the VCCC. Conditions include comparison of EC (red) to VSMC (green), (A, top); MEJ (red) to VSMC (green), (A, middle); and MEJ (red) to EC (green), (A, bottom). Arrows labeled 1–3 in A indicate three spots representing a unique protein with greater than 2.5 fold increase in fluorescent intensity (i.e., protein expression) in the MEJ versus VSMC and EC fractions. Using Quantitative DeCyder analysis, spots 1–3 are represented in a 3D visualization for each spot of interest and identified by a magenta tracer (Fig 2B), indicating protein fluorescent intensity peaks in VSMC, MEJ and EC fractions. The mass spectrum of spots 1 (C, top), 2 (C, middle) and 3 (C, bottom) identified each spot as plasminogen activator inhibitor-1. For all images, n=30 Tranwells.

Figure 3. Localization of PAI-1 at the MEJ

Immunoblots of VSMC, EC and MEJ fractions isolated from the VCCC blotted for PAI-1 and GAPDH as a loading control. Normalized quantification of protein expression for PAI-1 in each fraction is given in the adjacent histogram, n=4 (A). Immunocytochemistry of a single focal plane of a transverse section of the VCCC labeled for PAI-1 (green) and actin (red; phalloidin) demonstrate colocalized expression of both proteins regardless of the location in the Transwell pores (B). In vivo, the expression of PAI-1 on actin bridges that form between EC and VSMC (i.e. MEJs) in mesentery, cremaster and coronary microvascular beds is quantified using confocal microscopy in both C57Bl/6 and PAI-1^−/− mice, n=3 mice per experimental paradigm, 5 images per mouse (C). A representative TEM image of a MEJ from a mouse coronary arteriole labeled for PAI-1 with 10 nm gold particles is shown and quantified as number of beads per micrometer squared in D, n=3 mice per experimental paradigm, 5 images per mouse. Enlargement of white box in B is shown on right. In C, arrow indicates PAI-1 labeling. In D, “L” indicates lumen. Enlargement of red box insert in D is shown on right. Bar in B is 5 µm, bar in D is 0.5 µm, * p<0.05.

Figure 4. Effects of PAI-1 on MEJ formation in vitro

Metamorph analysis of changes in the number of MEJs per micrometer following inhibition of PAI-1 activity by application of 10 µg/mL PAI-1 specific mAb to the EC, EC and VSMC or VSMC monolayers is shown in A. Analysis of changes in the number of MEJs per micrometer following increases in PAI-1 activity by application of 0.1 µg/mL rPAI-1 to the EC, EC and VSMC or VSMC monolayers is shown in B. * p<0.05. For each condition (A–B), n=7 Transwells per condition, 10 images per Transwell.

Figure 5. Effects of PAI-1 on MEJ formation in vivo

Ultrastructural TEM images of coronary arterioles at low magnification (top) and higher magnification (bottom) from C57Bl/6 (A), PAI-1^−/− hearts (B) and C57Bl/6 fed a high fat western diet (C). TEM was used to visualize MEJs in coronary arterioles for each of the following experimental paradigms: C57Bl/6 hearts transplanted into recipient C57Bl/6 mice (D), PAI-1^−/− hearts transplanted into recipient PAI-1^−/− mice (E) and PAI-1^−/− hearts transplanted into recipient C57Bl/6 mice (F). TEM analysis of coronary arterioles isolated from saline injected PAI-1^−/− mice (G) and rPAI-1 injected PAI-1^−/− mice (E) are also shown. The number of MEJS per 10µm radially is quantified for images A-H in (I). Bar in each low magnification image (top image) is 2 µm, bar in A (higher magnification, top) is 2 µm and representative for all higher magnification images. In all images, * denotes vessel lumen and for each image, the lumen is located above the EC monolayer. EC and VSMC monolayers are separated by IEL for all images, arrows indicate MEJ in each magnified image. In I, *p<0.05 when compared to C57 animals. For images A–F,H, n=3 mice per experimental paradigm, 5 images per mouse. In G, n=2 mice, 5 images per mouse.

Figure 6. Uptake and expression of biotin-conjugated rPAI-1 at the MEJ

Immunoblots of VSMC, MEJ and EC fractions isolated from the VCCC stained with streptavidin. Three experimental paradigms were tested: no application of biotin-conjugated rPAI-1, application of biotin-conjugated rPAI-1 (0.1µg) to the VSMC monolayer and application of biotin-conjugated rPAI-1 (0.1µg) to only the EC monolayer. GAPDH is shown directly below each condition (A). Normalized quantification of protein expression for biotin-conjugated rPAI-1 for each fraction and experimental paradigm is given in (B). Immunocytochemistry on transverse sections of the VCCC for biotin-conjugated rPAI-1 using streptavidin (green) and F-actin (with phalloidin, red) for the following experimental paradigms: no application of biotin-conjugated rPAI-1, application of biotin-conjugated rPAI-1 (0.1µg) to the VSMC monolayer and application of biotin-conjugated rPAI-1 (0.1µg) to only the EC monolayer). For all images, VCCC were treated 30 minutes prior to isolation. Bar in C is 10 µm and representative for images (C-D). *p<0.05. For A–B, n=4.

Figure 7. Heterocellular Ca²⁺ communication in vascular cell co-cultures with increases and decreases in PAI-1 activity

Schematic representing the setup for measuring EC [Ca²⁺]_i following PE stimulation of the VSMC (A). The maximum values of EC [Ca²⁺]_i following PE stimulation is quantified and conditions include application of PAI-1 (+ rPAI-1); application of rPAI-1 + glycyrrhetinic acid (+ rPAI-1 + GA); control; control +GA, decreases in PAI-1 using a mAb against PAI-1, (−PAI-1); decreases in PAI-1 + glycerrhitinic acid (−PAI-1 + GA); and no MEJs (B). In C the temporal change in EC [Ca²⁺]_i after stimulation of VSMC with PE following application of rPAI-1 (+ rPAI-1), control; decreases in (− PAI-1); and no MEJs are shown. The time required to reach maximum EC [Ca²⁺]_i fluorescent intensity after VSMC stimulation with PE is shown for each condition: application of rPAI-1 (+ rPAI); control; and decreases in PAI-1 (−PAI-1) (D). For all images, VCCC were treated for the final 48 hours of culture, *p<0.05. n=3 Transwells per experimental condition.