1. Novel Small Leucine-Rich Repeat Protein Podocan Is a Negative Regulator of Migration and Proliferation of Smooth Muscle Cells, Modulates Neointima Formation, and Is Expressed in Human AtheromaClinical Perspective
by Randolph Hutter, Li Huang, Walter S. Speidl, Chiara Giannarelli, Paul Trubin, Gerhard Bauriedel, Mary E. Klotman, Valentin Fuster, Juan J. Badimon, and Paul E. Klotman
Circulation
Volume 128(22):
November 26, 2013
Copyright © American Heart Association, Inc. All rights reserved.

2. A–F, Effect of injury and podocan genotype on arterial podocan expression.
A–F, Effect of injury and podocan genotype on arterial podocan expression. A and D, Podocan was not detected in noninjured wild-type femoral arteries. Magnification, ×200 and ×1000; scale bar, 50 μm. B and E, After injury, brown labeling (arrows) for podocan was clearly seen in media and neointima of wild-type arteries at 4 weeks. Magnification, ×200 and ×1000; scale bar, 50 μm. C and F, After injury, an artery with podocan-deficient genotype exhibited a large neointima and showed, as expected, no podocan labeling, serving as negative control for podocan immunostaining. Magnification, ×200 and ×1000; scale bar, 50 μm. G–L, Time course of podocan expression after injury in wild-type artery. G and H, At 1 week after injury, podocan was hardly detectable in the media, whereas α-actin was expressed strongly. Magnification, ×200; scale bar, 50 μm. I and J, At 2 weeks, much stronger, albeit patchy, medial podocan expression appeared (arrows). Of note, podocan was also seen in early neointima. Magnification, ×200; scale bar, 50 μm. K and L, At 4 weeks, podocan expression (arrows) remained strong in neointima and media in close spatial association with α-actin signals. Magnification, ×200; scale bar, 50 μm. me indicates media; ni, neointima; and WT, wild type.
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

3. Time course of arterial response to injury with wild-type and podocan-deficient genotype.
Time course of arterial response to injury with wild-type and podocan-deficient genotype. Combined Masson-elastin staining: Wild-type genotype (A–C) and podocan-deficient genotype (D–F). A and D, At 1 week, cells adhered along the arterial surface on the luminal side of the media (me), and an adventitial cellular infiltrate formed in both groups in a similar fashion. Magnification, ×200; scale bar, 50 μm. B and E, At 2 weeks, a comparably sized typical early neointimal lesion (ni) with densely packed cells had formed in both groups. Magnification, ×200; scale bar, 50 μm. C and F, At 4 weeks, a moderately sized arterial lesion (ni) had formed with wild-type genotype; in contrast, with podocan-deficient genotype, the neointima showed a strongly increased size. Magnification, ×200; scale bar, 50 μm. M, Comparison of neointima formation with wild-type and podocan-deficient genotype; neointima area in ×10−2 mm2 (independent-sample t test). Smooth muscle α-actin: Wild-type genotype (G–I) and podocan-deficient genotype (J–L). G and J, At 1 week, α-actin expression (brown labeling) was predominantly seen in the media (me) in both groups; no neointima had formed yet. Magnification, ×200. H and K, At 2 weeks, nascent neointimal (ni) α-actin expression and a trend toward higher numbers of smooth muscle cells with the podocan-deficient genotype could be seen. Magnification, ×200; scale bar, 50 μm. I and L, At 4 weeks, a steep increase in the number of α-actin positive cells was observed in neointima (ni) with podocan-deficient genotype. Magnification, ×200; scale bar, 50 μm. N, Comparison of neointimal smooth muscle cell (SMC) density with wild-type and podocan-deficient genotype: cell density in ×103 mm2 (independent-sample t test).
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

4. Arterial proliferation with wild-type and podocan-deficient genotype.
Arterial proliferation with wild-type and podocan-deficient genotype. Ki-67 (FITC) and smooth muscle α-actin (Texas Red) double labeling: WT genotype (A–F) and podocan-deficient genotype (G–L). A and G, At 1 week, early after injury, Ki-67-positive (green) and α-actin–positive (red) smooth muscle cells (arrow) were seen in the media (me) in both groups. Magnification, ×200; scale bar, 50 μm. D and J represent matching IgG-isotype control stainings. B and H, At 2 weeks, only a few Ki-67 signals (arrows) were seen in both groups, consistent with a gradual decline in proliferation after the first week. Magnification, ×200; scale bar, 50 μm. E and K were negative controls. C and I, an unusually late rise in proliferation of smooth muscle cells (red α-actin labeling) was detected at 4 weeks by nuclear Ki-67 (green) labeling (arrows) with podocan-deficient genotype. Magnification, ×200; scale bar, 50 μm. F and L were negative controls. M, Comparison of Ki-67 expression with WT and podocan-deficient genotype after arterial injury, expressed as percentage of cells (independent-sample t test).
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

5. Late arterial proliferation with wild-type and podocan-deficient genotype.
Late arterial proliferation with wild-type and podocan-deficient genotype. A, D, and G, Wild-type genotype; B, E, and H, podocan-deficient genotype. A–C, Hematoxylin-and-eosin staining. Compared with wild-type (A), the strong increase in neointima (ni) with podocan-deficient genotype (B) persisted even at 6 weeks. Noninjured artery (C) of podocan-deficient mouse served as negative control for proliferation labeling. Magnification, ×200; scale bar, 50 μm. D–F, Ki-67 and smooth muscle α-actin double labeling. D, At 6 weeks, no green (FITC) Ki-67 labeling was seen in neointima (ni) or media (me) in wild-type genotype. E, With podocan-deficient genotype, however, green nuclear Ki-67 labeling (arrows) was seen in neointima (ni) even 6 weeks after injury. F, Noninjured podocan-deficient contralateral artery did not show any ki-67 signal. Magnification, ×200; scale bar, 50 μm. G–I and K, Labeling with bromodeoxyuridine (BrdU). G, In smaller wild-type neointima (ni), BrdU labeling was absent at 6 weeks after injury. H, In contrast, multiple nuclei with brown labeling (arrows) indicating BrdU incorporation were seen in hypercellular neointima with podocan-deficient genotype. Magnification, ×200; scale bar, 50 μm. I, Brown BrdU labeling (arrows) was also seen in bone marrow (bm) and served as positive control. K, Brown labeling was absent with isotype control staining in bone marrow. Magnification, ×400; scale bar, 50 μm. J, Comparison of Ki-67 and BrdU expression with wild-type and podocan-deficient genotype 6 weeks after arterial injury, expressed as percentage of cells (independent-sample t test).
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

6. Effect of wild-type and podocan-deficient genotype on murine aortic smooth muscle cells (SMC).
Effect of wild-type and podocan-deficient genotype on murine aortic smooth muscle cells (SMC). A and B, Aortic explant culture. A, The edge of wild-type aortic explant showed no SMC outgrowth at day 3. Magnification, ×400; scale bar, 50 μm. B, In contrast, numerous SMCs were seen at the edge of a podocan-deficient aortic explant at the same time point. Magnification, ×400; scale bar, 50 μm. C–E, SMC migration and proliferation. C, SMC migration was increased with podocan-deficient genotype compared with wild-type in a colorimetric test based on the Boyden chamber principle (independent-sample t test). D, Podocan-deficient SMCs also grew faster as measured by the MTS assay (independent-sample t test). E, Podocan-deficient SMCs transfected with podocan vector slowed their growth to wild-type-level (independent-sample t test). F–H, Wnt-TCF pathway activation. F, In SMCs with podocan-deficient genotype, the ratio of phosphorylated to nonphosphorylated β-catenin was reversed as seen by Western blot. G, Transcriptional activity of Wnt-TCF pathway measured directly by TOPflash/FOPflash assay was also increased with podocan-deficient genotype. H, Podocan-deficient SMCs treated with small inhibitory RNAs to β-catenin showed inhibition of growth. I–L, Effect of podocan overexpression on human aortic SMCs. I, Western blot confirmed overexpression of the human form of podocan. Podocan in control and empty vector–treated SMCs was below the detection threshold. J, SMC migration was reduced by 29% with podocan overexpression. K, SMC proliferation was reduced by 32% with podocan overexpression. L, SMC-overexpressing podocan showed an increase in phosphorylated β-catenin on Western blot compared with nontreated or empty vector–treated SMCs, which indicates Wnt-TCF pathway suppression. BRDU indicates bromodeoxyuridine; eGFP, enhanced green fluorescent protein; OD 490, optical density 490 nm; OD 588, optical density 588 nm; PDGF, platelet-derived growth factor; and siRNA, small inhibitory RNA.
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

7. Effect of wild-type and podocan-deficient genotype on Wnt pathway after arterial injury. α-actin (Texas Red) and nonphosphorylated β-catenin (FITC) double labeling: A and D, wild-type genotype; B, C, E, F, and G–K, podocan-deficient genotype.
Effect of wild-type and podocan-deficient genotype on Wnt pathway after arterial injury. α-actin (Texas Red) and nonphosphorylated β-catenin (FITC) double labeling: A and D, wild-type genotype; B, C, E, F, and G–K, podocan-deficient genotype. A–F, Low-power magnification. An antibody specific for the nonphosphorylated form of β-catenin gave a much stronger green (FITC) signal in neointima (ni) with podocan-deficient genotype (B and E) than with wild-type genotype (A and D). Magnification, ×200; scale bar, 50 μm. C and F, Matching isotype controls are shown for podocan-deficient neointima. G–K, High-power magnification. Comparison of single DAPI (G), single nonphosphorylated β-catenin labeling (H), and both combined (I) under high-power magnification clearly demonstrated the nuclear location of β-catenin signals in podocan-deficient neointima, indicating β-catenin nuclear translocation, a hallmark of true Wnt pathway activation. Magnification, ×1000; scale bar, 50 μm. These cells also stained positive for α-actin (J), which identified them as smooth muscle cells, and showed colocalization with nonphosphorylated β-catenin signals (K). L, Comparison of nonphosphorylated β-catenin expression in neointima with wild-type and podocan-deficient genotype after arterial injury, expressed as percentage of cells (independent-sample t test). me indicates media; ni, neointima; and NPBC, nonphosphorylated β-catenin.
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

8. Expression of podocan in human atheroma: Primary carotid atheroma (A–D), primary coronary lesion (E, F, I, J, M, N, Q, R, U, and V), and restenotic coronary lesion (G, H, K, L, O, P, S,T, W, and X).
Expression of podocan in human atheroma: Primary carotid atheroma (A–D), primary coronary lesion (E, F, I, J, M, N, Q, R, U, and V), and restenotic coronary lesion (G, H, K, L, O, P, S,T, W, and X). A–D, Podocan immunostaining. An antibody specific for the human form of podocan yielded strong brown labeling in the intima of carotid atheroma (A and C). Magnification, ×100; scale bar, 50 μm. Matching isotype staining on adjacent section showed no labeling (B and D). E–G, Combined Masson-elastin (CME) staining. Comparison of the histoarchitecture of primary and restenotic coronary lesions showed distinct differences. E, Spindle-shaped smooth muscle cells were surrounded by large spaces of extracellular matrix at a rather low cell density in primary lesions. G, In restenotic tissue, abundant numbers of smooth muscle cells were tightly clustered and surrounded by a comparatively smaller extracellular matrix space. Magnification, ×50, ×100, and ×200; scale bar, 50 μm. F–V, Two versions of podocan and smooth muscle cell double labeling. F and H, Smooth muscle α-actin (FITC) and podocan (Texas Red) double labeling. Low-power magnification images revealed the inverse relation between the degree of intimal podocan labeling (red) and the density of intimal smooth muscle cells (green) in primary (F) compared with restenotic (H) coronary plaque tissue. J, M, N, R, U, and V, Smooth muscle α-actin (Texas Red) and podocan (FITC) double labeling. J, M, N, R, U, and V, With reversed double labeling, higher-power magnification images confirmed that large extracellular matrix spaces in primary lesions surrounding the red-labeled smooth muscle cells were enriched with podocan, as shown by extensive green labeling (arrows). L, O, P, T, W, and X, In contrast, green podocan labeling (arrows) in restenotic tissue covered a much smaller area and was restricted to the immediate vicinity of red-labeled smooth muscle cells. Magnification, ×200 and ×1000; scale bar, 50 μm. I, Q, K, and S, Corresponding intimal locations in adjacent serial sections were also shown by light microscopy and CME staining. Magnification, ×200 and ×1000; scale bar, 50 μm. Of note, in both lesion types, staining with an isotype control antibody that matched the podocan antibody did not yield any green signals (z, I–IV). Y, Comparison of podocan expression (% area) and smooth muscle cell density (cells per mm2) in primary and restenotic coronary lesions; (independent-sample t test).
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.

9. Activation of Wnt-TCF pathway in human atheroma: primary coronary lesion (A, C, and E) and restenotic coronary lesion (B, D, F, H, and I). α-actin (Texas Red) and nonphosphorylated β-catenin (FITC) double labeling.
Activation of Wnt-TCF pathway in human atheroma: primary coronary lesion (A, C, and E) and restenotic coronary lesion (B, D, F, H, and I). α-actin (Texas Red) and nonphosphorylated β-catenin (FITC) double labeling. An antibody specific for the nonphosphorylated form of β-catenin yielded strong green (FITC) signals in intimal cells of a restenotic coronary lesion (F), whereas no signals were seen in the intima of a primary lesion (E). Magnification, ×400; scale bar, 50 μm. These cells also had typical smooth muscle cell morphology (B) and stained positive for α-actin (D). Magnification, ×400; scale bar, 50 μm. H and I, High-power magnification. Comparison of single nonphosphorylated β-catenin labeling (H) and combined labeling with DAPI (I) clearly demonstrated the nuclear location of β-catenin signals in restenotic intima, indicating β-catenin nuclear translocation, a hallmark of true Wnt-TCF pathway activation. Magnification, ×1000; scale bar, 50 μm. G, Comparison of nonphosphorylated β-catenin expression in the intima of primary and restenotic coronary lesions, expressed as percentage of cells (independent-sample t test). NP-BC indicates nonphosphorylated β-catenin.
Randolph Hutter et al. Circulation. 2013;128:
Copyright © American Heart Association, Inc. All rights reserved.