<strong>Rescue of murine hind limb ischemia via angiogenesis and lymphangiogenesis promoted by cellular communication network factor 2</strong>

<strong>Figure 1. Study design.</strong> <strong>a. </strong>Study design. Twelve-week-old male C57BL6/J mice with induced hind limb ischemia received an injection of CCN2 15 ng/PBS 100 μL (CCN2 group) or PBS 100 μL alone (control group). Blood flow in the hind limb was monitored by laser Doppler on postoperative days 0, 1, 3, 7, 14, and 28 (green triangles). Samples for RT-PCR, ELISA, and immunofluorescent staining were obtained from some mice on the scheduled days (blue and red triangles). <strong>b</strong>. Diagram showing injection of CCN2 into the thigh muscles of mice with induced hind limb ischemia. CCN2, cellular communication network factor 2; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT-PCR, real-time polymerase chain reaction <strong>Figure 2. Blood flow in ischemic hind limbs after treatment with CCN2.</strong> <strong>a</strong>. Representative laser Doppler images of mice in the control and CCN2 groups on postoperative days 0, 1, 3, 7, 14, and 28. Blood flow is visualized and differentiated by the colors red (rich blood flow) and blue (poor blood flow). The white triangle in the figure on postoperative day 7 in the CCN2 group indicates recovery of blood flow. <strong>b.</strong> Change in the ischemic/non-ischemic ratio in the control group (n=10) and the CCN2 group (n=11). The data are expressed as the mean ± standard error. *P&lt;.05; **P&lt;.01; ***P&lt;.001. CCN2, cellular communication network factor 2; POD, postoperative day <strong>Figure 3. Blood vessels in the ischemic hind limb after treatment with CCN2.</strong> <strong>a.</strong> von Willebrand factor (vWF)-positive capillaries (blood vessels) in myofibers of the ischemic hind limb in the control group (left) and the CCN2 group (right). The capillaries (stained in red) are indicated by white triangles. Original magnification, ×400; scale bar, 50 µm. <strong>b.</strong> Change in density of vWF-positive capillaries per myofiber in the control group (n=5; gray) and the CCN2 group (n=5; light blue). The data are expressed as the mean ± standard error. *P&lt;.05; **P&lt;.01; ***P&lt;.001; ****P&lt;.0001. CCN2, cellular communication network factor 2; POD, postoperative day <strong>Figure 4. Lymphatic vessels in the ischemic hind limb after treatment with CCN2.</strong> <strong>a.</strong> Podoplanin-positive capillaries (lymphatic vessels) in myofibers of the ischemic hind limb in the control group (left) and the CCN2 group (right). The capillaries (stained in red) are indicated by white triangles. Original magnification, ×400; scale bar, 50 µm. <strong>b.</strong> Change in density of podoplanin-positive capillaries per myofiber in the control group (n=5; gray) and the CCN2 group (n=5; light blue). The data are expressed as the mean ± standard error. *P&lt;.05; **P&lt;.01; ***P&lt;.001; ****P&lt;.0001. CCN2, cellular communication network factor 2; POD, postoperative day <strong>Figure 5. Gene expression and protein levels of angiogenesis and lymphangiogenesis markers in the thigh muscles after treatment with CCN2.</strong> <strong>a.</strong> <em>Vegfa</em> (left) and <em>Vegfc</em> (right) expression levels in the ischemic hind limb on postoperative days 3 and 7 in the control group (gray) and the CCN2 group (light blue), n=7–8 per group. <strong>b.</strong> VEGF-A (left) and VEGF-C (right) levels in the ischemic hind limb on postoperative days 3 and 7 in the control group (gray) and the CCN2 group (light blue), n=6–7 per group. The data are expressed as the mean ± standard error, n=6–7 per group. *P&lt;.05; **P&lt;.01; ***P&lt;.001. ns; not significant. CCN2, cellular communication network factor 2; POD, postoperative day; VEGF, vascular endothelial growth factor <strong>Figure 6. Effects of CCN2 on proliferation of VECs and LECs and lymphangiogenesis-correlated gene expression under hypoxia.</strong> The number of murine VECs (passage 6, n=5; <strong>a</strong>) and LECs (passage 6, n=5; <strong>b</strong>) cultured with and without CCN2 (CCN2 group and control group, respectively) under hypoxia after 0, 6, 12, 24, and 48 h of incubation. Cellular proliferation was assessed by the MTS assay. The data are expressed as the mean ± standard error. *P&lt;.05; **P&lt;.01; ***P&lt;.001. ns; not significant. <strong>c-f.</strong> Changes in expression of <em>Vegfc</em> (<strong>c</strong>), <em>Vegfr3</em> (<strong>d</strong>), <em>Tgfβ1</em> (<strong>e</strong>), and <em>Hif1α</em> (<strong>f</strong>) in LECs cultured with and without CCN2 (CCN2 group and control group, respectively) under hypoxia. <em>Gapdh</em> was used as an internal control, and gene expression levels were expressed relative to <em>Gapdh</em> mRNA. The data are expressed as the mean ± standard error, n=3/group. *P&lt;.05; **P&lt;.01; ***P&lt;.001. CCN2, cellular communication network factor 2; LECs, lymphatic endothelial cells; VECs, vascular endothelial cells <strong>Figure 7. Influence of </strong><em><strong>Tgfβ1</strong></em><strong> knockdown on lymphangiogenesis in LECs under hypoxia.</strong> <em>Tgfb1</em> knockdown were performed by transfection with siRNA against <em>Tgfb1</em> using two types of <em>Tgfb1</em> siRNA (siRNA#1 and siRNA#2).<strong> a.</strong> Expression level of <em>Tgfβ1</em> <em>i</em>n LECs after administration of CCN2 under hypoxia. The data are expressed as the mean ± standard error, n=3/group. <strong>b.</strong> Cellular proliferation of LECs after administration of CCN2 under hypoxia. The data are expressed as the mean ± standard error. The LECs were in passage 6, n=5/group. <strong>c.</strong> Levels of ERK 1/2, SMAD 2, SMAD 3, and SMAD 4 in LECs after administration of CCN2 under hypoxia. The median fluorescence intensity of these proteins was measured using the Luminex®︎ system, n=3/group. <strong>d.</strong> Expression levels of <em>Vegfc</em> and <em>Vegfr3</em> in LECs after administration of CCN2 under hypoxia. The data are expressed as the mean ± standard error, n=3/group. *P&lt;.05; **P&lt;.01; ***P&lt;.001. ns; not significant. CCN2, cellular communication network factor 2; ERK, extracellular signal-regulated kinase; LECs, lymphatic endothelial cells; SMAD, suppressor of mothers against decapentaplegic