Therapeutic Angiogenesis for Treating Cardiovascular Diseases

<p>Cardiovascular disease is the leading cause of death worldwide and is often associated with partial or full occlusion of the blood vessel network in the affected organs. Restoring blood supply is critical for the successful treatment of cardiovascular diseases. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing vessels. In this review, we discuss strategies developed for therapeutic angiogenesis using single or combinations of biological signals, cells and polymeric biomaterials. Compared to direct delivery of growth factors or cells alone, polymeric biomaterials provide a three-dimensional drug-releasing depot that is capable of facilitating temporally and spatially controlled release. Biomimetic signals can also be incorporated into polymeric scaffolds to allow environmentally-responsive or cell-triggered release of biological signals for targeted angiogenesis. Recent progress in exploiting genetically engineered stem cells and endogenous cell homing mechanisms for therapeutic angiogenesis is also discussed.</p

Comparative analysis of gene expression identifies distinct molecular signatures of bone marrow- and periosteal-skeletal stem/progenitor cells

<div><p>Periosteum and bone marrow (BM) both contain skeletal stem/progenitor cells (SSCs) that participate in fracture repair. However, the functional difference and selective regulatory mechanisms of SSCs in different locations are unknown due to the lack of specific markers. Here, we report a comprehensive gene expression analysis of bone marrow SSCs (BM-SSCs), periosteal SSCs (P-SSCs), and more differentiated osteoprogenitors by using reporter mice expressing Interferon-inducible <i>Mx1</i> and <i>Nestin</i>^GFP, previously known SSC markers. We first defined that the BM-SSCs can be enriched by the combination of <i>Mx1</i> and <i>Nestin</i>^GFP expression, while endogenous P-SSCs can be isolated by positive selection of <i>Mx1</i>, CD105 and CD140a (known SSC markers) combined with the negative selection of CD45, CD31, and <i>osteocalcin</i>^GFP (a mature osteoblast marker). Comparative gene expression analysis with FACS-sorted BM-SSCs, P-SSCs, <i>Osterix</i>⁺ preosteoblasts, CD51⁺ stroma cells and CD45⁺ hematopoietic cells as controls revealed that BM-SSCs and P-SSCs have high similarity with few potential differences without statistical significance. We also found that CD51⁺ cells are highly heterogeneous and have little overlap with SSCs. This was further supported by the microarray cluster analysis, where the two SSC populations clustered together but are separate from the CD51⁺ cells. However, when comparing SSC population to controls, we found several genes that are uniquely upregulated in endogenous SSCs. Amongst these genes, we found KDR (aka Flk1 or VEGFR2) to be most interesting and discovered that it is highly and selectively expressed in P-SSCs. This finding suggests that endogenous P-SSCs are functionally very similar to BM-SSCs with undetectable significant differences in gene expression but there are distinct molecular signatures in P-SSCs, which can be useful to specify P-SSC subset <i>in vivo</i>.</p></div

Functional identification of P-SSCs and BM-SSCs.

<p><b>(A)</b> Interferon inducible <i>Mx1</i>^<i>+</i> SSCs (red) are shown to contribute to majority osteoblasts (green, overlap yellow) <i>in vivo</i>. <b>(B)</b> <i>Mx1</i>^<i>+</i> SSCs represent long-term osteolineage progenitor cells in BM and periosteal tissues. <i>In vivo</i> imaging shows that <i>Mx1</i>^<i>+</i> SSCs reside in bone marrow, suture, and periosteum of calvarial bones. Immunofluorescent staining of tibial metaphysis of Mx1/Tomato/Ocn-GFP mice shows that <i>Mx1</i>⁺ SSCs (red, arrows) are abundant in periosteum (PO) (Tibia IHC). <b>(C)</b> P-SSCs from periosteal tissues are FACS-sorted by CD45⁻CD31⁻Ter119⁻CD105⁺CD140a⁺ and <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻, which are referred to as <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs. <b>(D)</b> <i>Mx1</i>^<i>+</i><i>Nestin</i>^<i>+</i> BM-SSCs are perivascular cells in BM but are undetectable in periosteum and calvarial suture. <b>(E)</b> <i>Mx1</i>⁺<i>Nes</i>⁺ cells within CD45⁻CD31⁻Ter119⁻CD105⁺ CD140a⁺ SSC fraction in bone marrow are isolated by FACS-sorting and are referred to as <i>Mx1</i>⁺<i>Nes</i>⁺ BM-SSCs. Notably, CD105⁺CD140a⁺ progenitors are heterogeneous <i>Mx1</i>⁺ and <i>Nestin</i>⁺ cells.</p

Identification of differentially expressed genes between P-SSCs and BM-SSCs and controls.

<p><b>(A & B)</b> Differential gene expression between CD45^<i>+</i> cells and <i>Osx</i>^<i>+</i> cells with <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs (A) and <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (B). <b>(C)</b> Number of differentially expressed genes between SSC populations and controls shows 101 for <i>Mx1</i>^<i>+</i><i>Ocn</i>^<i>−</i> P-SSCs, 84 for <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs, and 55 overlapping genes. <b>(D)</b> List of genes that were upregulated in SSCs compared to controls includes Flt1 (VEGF receptor 1) and KDR (VEGF receptor 2), despite removal of CD31 endothelial lineage cells from these populations.</p

P-SSCs selectively express KDR.

<p><b>(A)</b> Higher surface expression of KDR and CD140a in P-SSCs (CD45⁻CD31⁻Ter119⁻<i>Mx1</i>^<i>+</i><i>Ocn</i>^<i>−</i>) compared to periosteal osteoblast controls (CD45⁻CD31⁻Ter119⁻<i>Mx1</i>^<i>-</i><i>Ocn</i>^<i>+</i>) from <i>Mx1</i>^<i>Cre</i><i>; Rosa</i>^{<i>Tomato</i>}; <i>Osteocalcin</i>^<i>GFP</i> mice. <b>(B)</b> Undetectable expression of KDR in <i>Mx1</i>^<i>+</i> BMSCs (CD45⁻CD31⁻Ter119⁻<i>Mx1</i>^<i>+</i><i>Ocn</i>^<i>−</i>) in the same mice (<i>Mx1</i>^<i>Cre</i><i>; Rosa</i>^{<i>Tomato</i>}; <i>Osteocalcin</i>^<i>GFP</i>). <b>(C)</b> KDR⁺CD140a⁺ FACS analysis of BM-SSCs (CD45⁻CD31⁻Ter119⁻<i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i>) from <i>Mx1</i>^<i>Cre</i>; <i>Rosa26</i>^{<i>Tomato</i>}; <i>Nestin</i>^<i>GFP</i> transgenic mice similarly shows decreased expression of KDR in BM-SSCs. <b>(D)</b> Summary of FACS analysis demonstrates that <i>Mx1</i>^<i>+</i><i>Ocn</i>^<i>−</i> P-SSCs uniquely express KDR and CD140a (77%) compared to BM-SSCs and control populations (n = 3, p < 0.0001). <b>(E)</b> Confirmation of the selective expression of KDR and CD140a in <i>Prx1</i> ^GFP<i>+</i> P-SSCs.</p

Commonly used markers for BM-SSCs yield a heterogeneous mixture, but are similar to P-SSCs.

<p><b>(A-C)</b> Scatter plot comparison between <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs (A), <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (B), and CD51^<i>+</i> BMSCs (C) with CD45^<i>+</i> cells, demonstrates that these populations are likewise different from CD45^<i>+</i> hematopoietic cells from the BM compartment. <b>(D-F)</b> Scatter plot comparison between <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs (D), <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (E), and CD51^<i>+</i> BMSCs (F) with <i>Osx</i>^<i>+</i> osteoprogenitor cells shows that each of these populations are more functionally similar to the osteolineage cells. <b>(G)</b> Direct comparison between CD51^<i>+</i> BMSCs and <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs demonstrates that these two commonly used selection markers for BM-SSCs yield a heterogeneous mixture of cells. <b>(H)</b> <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs and <i>Nes</i>^<i>+</i> cells are essentially the same population of cells. <b>(I & J)</b> Comparing <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs with CD51^<i>+</i> BMSCs (I) shows that these are functionally different cell-populations, but comparison with <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (J) shows few differences. <b>(K)</b> Cluster analysis of these cell populations confirms scatter plot analysis and shows that <i>Mx1</i>^<i>+</i><i>Ocn</i>⁻ P-SSCs and <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs cluster together, but each of these populations are distinct from CD51^<i>+</i> BMSCs (<i>p</i> < 0.05).</p

P-SSCs and BM-SSCs express common SSC markers.

<p><b>(A)</b> Gene commons analysis shows that <i>Mx1</i>⁺<i>Ocn</i>⁻ P-SSCs (<i>MON</i>) and <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (<i>MNS</i>) highly express Leptin receptor (LepR) and Gremlin 1, demonstrating that these SSC populations share characteristics with previously studied early postnatal SSC and progenitor populations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190909#pone.0190909.ref029" target="_blank">29</a>]. Further, KDR is found to be uniquely expressed in P-SSCs compared to other SSCs. <b>(B)</b> Hematopoietic cells (CD45⁺), osteoblasts (OcnG⁺), <i>Mx1</i>^<i>+</i><i>Nes</i>^<i>+</i> BM-SSCs (<i>MNS</i>) and <i>Mx1</i>⁺ P-SSCs were sorted, and the levels of the indicated genes were quantified by qPCR.</p