αSMA expression is upregulated in cav-1 -/- mammary glands.

<p><b>(A)</b> Immunohistochemistry of αSMA in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated that αSMA expressing cells are predominantly localized to the myoepithelial compartments in cav-1 +/+ and cav-1 -/- glands. αSMA expressing cells are also observed in adipose stromal cells (arrows) in cav-1 +/+ and cav-1 -/- glands <b>(B)</b> The percent expression of αSMA was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. <b>(C)</b> The intensity of αSMA staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05. Results are reported as the mean ± the SEM.</p

Proliferation of ductal epithelial cells is increased in cav-1 -/- glands.

<p><b>(A)</b> Immunohistochemistry of ki67 cells in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results from representative images demonstrated that ki67 expressing ductal epithelial cells (arrows) are markedly increased in cav-1 -/- glands in comparison to cav-1 +/+ glands. <b>(B)</b> The average number of ki67 positive cells was significantly higher in cav-1 -/- glands in comparison to cav-1 +/+ glands. Scale bars are 20uM. **p≤0.01. Results are reported as the mean ± the SD.</p

Collagen I expression and organization are upregulated in cav-1 -/- mammary glands.

<p><b>(A)</b> PSR staining for collagen I in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Imaging demonstrates extensive expression of collagen I in the adipose stroma and in the peri-ductal region of cav-1 -/- glands. The collagen I fibers appear dense and tightly packed in cav-1 -/- glands. In contrast, overall collagen I expression is considerably lower in cav-1 +/+ glands with fibers being predominantly located to the peri-ductal region. Here, fibers are less packed and distinct fibrils are apparent. <b>(B)</b> The percent expression of PSR stained collagen I was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. <b>(C)</b> The intensity of PSR stained collagen I was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. <b>(D)</b> Representative SHG images show an abundance of dense, thick collagen fibers in cav-1 -/- glands. Collagen fibers and fiber bundles also appear more organized and aligned (red arrows). Cav-1 +/+ glands express comparably less collagen which presents as thin, unorganized fibers. Scale bars are 25 μM. Col: collagen. *p≤0.05, **p≤0.01. Results are reported as the mean ± the SEM.</p

Ductal architecture is disrupted in cav-1 -/- glands.

<p><b>(A)</b> The number of ducts were enumerated in αSMA stained sections from cav-1 -/- and cav-1 +/+ glands. Results demonstrated a highly significant average number of ducts per image in cav-1 -/- glands in comparison to cav-1 +/+ glands. <b>(B)</b> Images of fibronectin stained sections illustrate the abundance of large, elongated, branched ducts (red arrows) in cav-1 -/- glands. Ducts in cav-1 +/+ glands appear small and relatively round (red arrows). Scale bars are 100μM. <b>(C)</b> H&E stained images of glands demonstrate the presence of stacked ductal epithelial cells in cav-1 -/- sections, indicative of ductal hyperplasia. Cav-1 +/+ glands exhibit a continuous monolayer of ductal epithelial cells. Scale bars are 20μM. <b>(D)</b> The ductal circumference, measured as the region immediately outside the myoepithelial compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal circumference was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. <b>(E)</b> The area, measured as the region between the myoepithelial and luminal compartment, was analyzed in αSMA stained sections in cav-1 -/- and cav-1 +/+ glands. The ductal area was significantly greater in cav-1 -/- as opposed to cav-1 +/+ glands. <b>(F)</b> This image of an αSMA stained cav-1 +/+ duct illustrates the vectors used to assign stromal and lumen outlines used to analyze ductal measurements. The red line shows the stromal border while the blue line shows the lumen border. Scale bar is 50uM. *p≤0.05, **p≤0.01, ***p≤0.001. Results are reported as the mean ± the SEM.</p

Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands

<div><p>The extracellular matrix (ECM) is abnormal in breast tumors and has been reported to contribute to breast tumor progression. One factor, which may drive ongoing matrix synthesis in breast tumors, is the loss of stromal caveolin-1 (cav-1), a scaffolding protein of caveolae, which has been linked to breast tumor aggressiveness. To determine whether loss of cav-1 results in the abnormal expression of matrix proteins, mammary glands from cav- 1-/- and cav- 1 +/+ mice were investigated for differences in expression of several ECM proteins. In addition, the presence of myofibroblasts, changes in the vessel density, and differences in duct number and size were assessed in the mammary glands of both animal models. Using immunohistochemistry, expression of fibronectin, tenascin-C, collagens and αSMA were significantly increased in the mammary glands of cav-1-/- mice. Second harmonic generation revealed more organized collagen fibers in cav-1 -/- glands and supported immunohistochemical analyses of increased collagen abundance in the glands of cav-1 -/- mice. Analysis of the ductal structure demonstrated a significant increase in the number of proliferating ducts in addition to significant increases in the duct circumference and area in cav-1 -/- glands compared to cav- 1 +/+ glands. Differences in microvessel density weren’t apparent between the animal models. In summary, we found that the loss of cav-1 resulted in increased ECM and α-SMA protein expression in murine mammary glands. Furthermore, we found that an abnormal ductal architecture accompanied the loss of cav-1. These data support a role for cav-1 in maintaining mammary gland structure.</p></div

Tenascin-C expression is upregulated in cav-1 -/- mammary glands.

<p><b>(A)</b> Immunohistochemistry of tenascin-C expression in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated an increased expression of tenascin-C in the peri-ductal and adipose stroma in cav-1 -/- glands. This is in contrast to cav-1 +/+ glands where tenascin-C expression is expressed in the peri-ductal stroma and to a lesser extent in the adipose stroma. Tenascin-C was also expressed in the stromal cells in cav- 1-/- and cav-1 +/+ glands (red arrows), albeit with a greater proportion of positive cells in cav-1 -/- glands. <b>(B)</b> The percent expression of tenascin-C was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. <b>(C)</b> The intensity of tenascin-C staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05, **p≤0.01. Tn-C: tenascin-C. Results are reported as the mean ± the SEM.</p

Fibronectin expression is upregulated in cav-1 -/- mammary glands.

<p><b>(A)</b> Immunohistochemistry of fibronectin expression in cav-1 +/+ and cav-1 -/- glands at low and high magnification. Results demonstrated an increased abundance fibronectin fibers surrounding the ducts and in the adipose stroma of cav-1 -/- glands. Fibronectin expression was also localized to the epithelial cells (red arrows) of the ducts in both cav-1 +/+ and cav-1 -/- glands. <b>(B)</b> The percent expression of fibronectin was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. <b>(C)</b> The intensity of fibronectin staining was significantly higher in cav-1 -/- glands compared to cav-1 +/+ glands. Scale bars are 50 μM and 20 μM in low and high magnification images, respectively. *p≤0.05. Fn: fibronectin. Results are reported as the mean ± the SEM.</p

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis

<div><p>The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α₅β₁ and α_vβ_3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.</p></div

Vascular kinetics is altered following culture of GFP+ ECs atop pUR4B or III-11C ECM.

<p><b>(A)</b> GFP+ ECs were seeded on Chambers, III-11C-ECM and pUR4B-ECM and were assessed for differences in vascular kinetics between hours 7 and 12 post-seeding. Graphs depict <b>(B)</b> total distance traveled, <b>(C)</b> net displacement, <b>(D)</b> average velocity, and <b>(E)</b> mean square displacement of the GFP+ ECs. *p≤0.05; **p≤0.01; ***p≤0.001. The error bars represent the SEM.</p

Additional file 8: of Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

Material 4, Cell Viability 20 and 30 μg, Cell viability analyses for HMFs encapsulated in 20 and 30 μg hydrogels. (XLSX 10 kb