i-Fect Delivers Again and Again

Silencing Lactate Dehydrogenase A in vivo

Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.

A mixture of our i-Fect^TM and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis. doi.org/10.1016/j.ebiom.2017.10.033.

Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.

The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disorders

Hypoxia Induced Angiogenesis and Tumor Microenvironment

Neuromic's HUVECS Used in Study

The tumor microenvironment is essential for promoting tumor physiology, structure, function, and growth. It is important that emerging therapies eradicate both tumors and related microenvironment. This important study focuses on the formation of these microenvironments: ERICA K. SCHNETTLER. THE FUNCTIONAL ROLE OF MIR-210 IN HYPOXIA-INDUCED ANGIOGENESIS. A DISSERTATION SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA. FEBRUARY 2017...Primary Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel, Switzerland) and Neuromics (Edina, MN)...

Figure: miR-210 sensitizes HUVECs to bFGF induced tube formation. (A)Representative images of HUVEC tube formation are shown at 10X magnification. Cells were treated with miR-210 or sc-miR, serum starved, and then plated on a Geltrex (reduced growth factor) layer in the presence or absence or bFGF to stimulate tube formation. (B) Histogram represents quantification of total tube length, number of nodes, and junctions in the tube formation assay described in A. Analysis done with ImageJ Angiogenesis Analyzer plugin.

We will continue to post our updates our primary Human Endothelial Cells as they develop.

Human Growth Factor+Neurotrophin+Cytokine Array-Test 40 Markers in on Assay

Sensitive, Specific and Cost Effective

Neuro-immuno and degenerative diseases and disorders commonly show dysregulation of Growth Factors and Cytokines. Using our Quantibody Neuroscience Arrays, we have measured the blood serum of clients with Neuro-inflammatory/immune response diseases/disorders including Autism Spectrum Disorder (ASD). Most showed lowered levels of Growth Factors/Neurotrophins and elevated levels of Inflammatory Response Cytokines.

In order to expand the number of biomarkers measured, we are pleased to announce the addition of our New Growth Factor+Neurotrophin+Cytokine Array. This will enable us to further determine the "finger prints" of these diseases/disorders at the protein level. This array includes: Immunogen: Amphiregulin, BDNF, bFGF, BMP-4, BMP-5, BMP-7, beta-NGF, EGF, EGFR, EG-VEGF (PK1), FGF-4,FGF-7 (KGF), GDF-15, GDNF, Growth Hormone, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-1, Insulin, M-CSF R, NGFR (TNFRSF16), NT-3, NT-4, Osteoprotegerin (TNFRSF11B), PDGF-AA, PLGF, SCF, SCF R (CD117/c-kit), TGF alpha, TGF beta 1, TGF beta 3, VEGF-A, VEGFR2, VEGFR3 and VEGF-D.

I will continue to post testing results here.

Using hESCs to Understand Vasculogenesis Processes

The Role of BMP4 Plays in Regulating Vascular Development

My friend, Dr. Steve Stice and team at UGA have published excellent findings on the process of vasculogenesis: N. L. Boyd, S. K. Dhara, R. Rekaya, E. A. Godbey, K. Hasneen, R. R. Rao, F. D. West III, B. A. Gerwe, S. L. Stice. BMP4 Promotes Formation of Primitive Vascular Networks in Human Embryonic Stem Cell–Derived Embryoid Bodies. Exp Biol Med (Maywood) June 2007 vol. 232 no. 6 833-843.
Highlights:
Human embryonic stem cells (hESC) have the capability to produce all of the cells of the body and have been used as in vitro models to study the molecular signals controlling differentiation and vessel assembly. One such regulatory molecule is bone morphogenetic protein-4 (BMP4), which is required for mesoderm formation and vascular/hematopoietic specification in several species. However, hESC grown in feeder-free conditions and treated with BMP4 differentiate into a cellular phenotype highly expressing a trophoblast gene profile. Therefore, it is unclear what role, if any, BMP4 plays in regulating vascular development in hESC. Here we show in two National Institutes of Health–registered hESC lines (BG02 and WA09) cultured on a 3D substrate of Matrigel in endothelial cell growth medium–2 that the addition of BMP4 (100 ng/ml) for 3 days significantly increases the formation and outgrowth of a network of cells reminiscent of capillary-like structures formed by mature endothelial cells (P < 0.05). Analysis of the expression of 45 genes by quantitative real time–polymerase chain reaction on a low-density array of the entire culture indicates a rapid and significant downregulation of pluripotent and most ectodermal markers with a general upregulation of endoderm, mesoderm, and endothelial markers. Of the genes assayed, BMPR2 and RUNX1 were differentially affected by exposure to BMP4 in both cell lines. Immunocytochemistry indicates the morphological structures formed were negative for the mature endothelial markers CD31 and CD146 as well as the neural marker SOX2, yet positive for the early vascular markers of endothelium (KDR, NESTIN) and smooth muscle cells (α-smooth muscle actin [αSMA]). Together, these data suggest BMP4 can enhance the formation and outgrowth of an immature vascular system.

Figures: Least-squared mean gene expression analysis of BMP4 versus control time course for BGO2 and WA09. BG02 (solid lines) and WA09 (dashed lines) were cultured for 3 days ± BMP4, then for another 5 days in EGM-2 only. Samples were collected on Days 0, 2, 4, 6, and 8; total RNA was extracted, and gene expression was analyzed by qRT-PCR. Least-squared mean comparison was then expressed as x-fold change with respect to Day 0 control. The gene expression time course for (A) OCT4, (B) HEY1, (C) GATA3, (D) NESTIN, (E) SOX2, (F) BMPR2, (G) KDR/Flk1, and (H) CD31/PECAM1 are shown. (BG02: −BMP4 = solid diamond; +BMP4 = solid square; WA09: −BMP4 = open triangle; +BMP4 = open circle; * P < 0.05 for BG02 only; ** P < 0.05 for WA09 only; *** P < 0.05 for both BG02 and WA09).http://ebm.sagepub.com/content/232/6/833.full

Images: Early vascular markers KDR, αSMA, and NESTIN are detected within the network structures and elsewhere. Cell cultures were dual-immunostained to detect co-expression of KDR with αSMA (A–C), KDR with NESTIN (D–F), or αSMA with NESTIN (G–I), and the nucleus was counterstained with DAPI. For each case, three different morphologic regions were represented: the residual EB (A, D, and G), the periphery of the EB (B, E, and H), and the thin network structures (C, F, and I). All images were acquired with a ×40 oil objective, and a Z series stack was projected into a single image. A color figure is available in the online version of the journal.

These findings confirm the use of stem cells and the EB as a model of early development including vasculogenesis (3, 4). Using hESC in this manner can provide insights into the mechanisms regulating the earliest events in human vasculogenesis. An understanding of how the vasculature is formed could also be applied to tissue engineering and angiogenic/ischemic therapies. Note: our Mouse Monoclonal Nestin Antibody is an excellent marker for early vasculogensis of the endothelium.

Opioid Receptor Antibodies Trifecta

This publication proposes a role for opioid receptors in treating cancers. It also references use of our μ, δ, and κ opioid receptor antibodies.

Kohei Yamamizu1, Sadayoshi Furuta, Shiori Katayama, Michiko Narita, Naoko Kuzumaki, Satoshi Imai, Hiroshi Nagase, Tsutomu Suzuki, Minoru Narita, and Jun K. Yamashita. The κ opioid system regulates endothelial cell differentiation and pathfinding in vascular development. Blood July 21, 2011 vol. 118 no. 3 775-785.

Highlights: The opioid system is, thus, a new regulator of vascular development that simultaneously modifies 2 distinct vascular properties, EC differentiation and vascular pathfinding. We confirmed that KOR, but not MOR, was highly expressed in various ECs such as HUVECs (data not shown), suggesting that KOR agonists could directly act on tumor ECs to suppress VEGF receptor expression, similar to the effects observed in embryonic ECs. If so, a combination therapy including an MOR agonist, morphine, and a KOR agonist (such as TRK820, a clinically approved drug in Japan for uremic pruritus) may prove useful for cancer therapy through the suppression of tumor angiogenesis by dual inhibition of VEGF ligands and receptors, extending the therapeutic benefits beyond pain relief.

Images: KOR was highly expressed in Flk1⁺ vascular progenitors. (A) RT-PCR showing mRNA expression of MOR, DOR, and KOR in ES cells, Flk1⁺ cells, cells after 1 or 3 days of Flk1⁺ cell culture (Flk-d1 or Flk-d3), CD31-positive cells (ECs) and CD31-negative cells (MCs) at Flk-d3. (B) Fluorescent staining for MOR, DOR, and KOR at Flk-d1. Nuclei are stained with DAPI (blue). Left, MOR; middle, DOR; right, KOR. Scale bars, 100 μm. (C) Double fluorescent staining for MOR, DOR, and KOR with CD31 (red) at Flk-d3. Nuclei are stained with DAPI (blue). Top, opioid (green) receptors (green); middle, CD31 (red); bottom, merged. Scale bars, 100 μm.

Angiogensis and CD antigens

We consider everything genesis as a core focus area. This includes embryogenesis, angiogenesis, neurogenesis and stem cell expansion and differentiation. This is important because there is an intersection between reagents and methods for studying embryo development and genesis pathways in "adult systems".

CD antigens serve as markers for growth and development. We are always on the look out for Customer Publications that reference use of these reagents in related applications. Here's one that just pinged our radar.

Karim Harhouri, Abdeldjalil Kebir, Benjamin Guillet, Alexandrine Foucault-Bertaud, Serge Voytenko, Marie-Dominique Piercecchi-Marti, Caroline Berenguer, Edouard Lamy, Frédéric Vely, Pascale Pisano, L'Houcine Ouafik, Florence Sabatier, José Sampol, Nathalie Bardin, Françoise Dignat-George, and Marcel Blot-Chabaud Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia. Blood, May 2010; 115: 3843 - 3851
Anti-rat antibodies used in this study are: anti-CD117 (Neuromics) and anti-CD146

Abstract: CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases

Image: c-KIT staining of rat skin (epidermis). c-KIT detection was done using anti-rabbit Cy3 conjugated antibodies (red color). DAPI was used to counterstain cell nuclei (blue color).Working dilution: 1:100-1:300.

Related Reagents:

• Immune Response Antibdodies


• Immune Response Proteins


• Cancer Research Antibodies


• Cancer Research Proteins


• Pain and Inflammation Research Antibodies


• Neurodegenerative Disease Antibodies


• Neurodegenerative Disease Proteins


• Apoptosis Research Reagents


• Stem Cell Research Reagents