Identification and Characterization of Novel Perivascular Adventitial Cells in the Whole Mount Mesenteric Branch Artery Using Immunofluorescent Staining and Scanning Confocal Microscopy Imaging
A novel perivascular adventitial cell termed, adventitial neuronal somata (ANNIES) expressing the neural cell adhesion molecule (NCAM) and the vasodilator neuropeptide, calcitonin gene-related peptide (CGRP), exists in the adult rat mesenteric branch artery (MBA) in situ. In addition, we have previously shown that ANNIES coexpress CGRP and NCAM. We now show that ANNIES express the neurite growth marker, growth associated protein-43(Gap-43), palladin, and the calcium sensing receptor (CaSR), that senses changes in extracellular Ca(2+) and participates in vasodilator mechanisms. Thus, a previously characterized vasodilator, calcium sensing autocrine/paracrine system, exists in the perivascular adventitia associated with neural-vascular interface. Images of the whole mount MBA segments were analyzed under scanning confocal microscopy. Confocal analysis showed that the Gap-43, CaSR, and palladin were present in ANNIES about 37 ± 4%, 94 ± 6%, and 80 ± 10% respectively, comparable to CGRP (100%). Immunoblots from MBA confirmed the presence of Gap-43 (48？kD), NCAM (120 and 140？kD), and palladin (90–92 and 140 kD). In summary, CGRP, and NCAM-containing neural cells in the perivascular adventitia also express palladin and CaSR, and coexpress Gap-43 which may participate in response to stress/injury and vasodilator mechanisms as part of a perivascular sensory neural network. 1. Introduction Vascular growth and remodeling occur in association with certain physiological and pathological conditions. In addition, vascular regeneration and repair are essential for the survival of blood vessels. These processes involve numerous cell types. There are still uncharacterized and less characterized cell types in vascular adventitia, which include vascular stem/progenitor cells [1–10] and adventitial neuronal somata (ANNIES) [11]. The vascular adventitia is a complicated tissue [12], which is found to be the most active layer in terms of cell turnover [13]. In addition, within the vascular adventitia resides amyelinated nerves known as “nerva vasorum” [14]. Using fluorescence confocal microscopy (FCM) to visualize vascular wall 3D organization of different cellular and extracellular elements of the intact artery with minimal 3D distortion [11, 13], we recently demonstrated ANNIES coexpressing neural cell adhesion molecule (NCAM) and calcitonin gene-related peptide (CGRP) in the adult rat mesenteric branch artery (MBA). These cells can be enzymatically dispersed and maintained in culture [11]. The present study was designed to further characterize ANNIES in adult rat MBA as
References
[1]
A. Pacilli and G. Pasquinelli, “Vascular wall resident progenitor cells. A review,” Experimental Cell Research, vol. 315, no. 6, pp. 901–914, 2009.
[2]
E. Zengin, F. Chalajour, U. M. Gehling et al., “Vascular wall resident progenitor cells: a source for postnatal vasculogenesis,” Development, vol. 133, no. 8, pp. 1543–1551, 2006.
[3]
C. W. Chen, E. Montelatici, M. Crisan et al., “Perivascular multi-lineage progenitor cells in human organs: regenerative units, cytokine sources or both?” Cytokine and Growth Factor Reviews, vol. 20, no. 5-6, pp. 429–434, 2009.
[4]
M. Corselli, C. W. Chen, M. Crisan, L. Lazzari, and B. Péault, “Perivascular ancestors of adult multipotent stem cells,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 30, no. 6, pp. 1104–1109, 2010.
[5]
M. Crisan, C. W. Chen, M. Corselli, G. Andriolo, L. Lazzari, and B. Péault, “Perivascular multipotent progenitor cells in human organs,” Annals of the New York Academy of Sciences, vol. 1176, pp. 118–123, 2009.
[6]
M. Crisan, B. Deasy, M. Gavina et al., “Purification and long-term culture of multipotent progenitor cells affiliated with the walls of human blood vessels: myoendothelial cells and pericytes,” Methods in Cell Biology, vol. 86, pp. 295–309, 2008.
[7]
M. Crisan, J. Huard, B. Zheng et al., “Purification and culture of human blood vessel-associated progenitor cells,” Current Protocols in Stem Cell Biology, chapter 2, pp. 2B.2.1–2B.2.13, 2008.
[8]
M. Crisan, S. Yap, L. Casteilla et al., “A perivascular origin for mesenchymal stem cells in multiple human organs,” Cell Stem Cell, vol. 3, no. 3, pp. 301–313, 2008.
[9]
D. Klein, H. P. Hohn, V. Kleff, D. Tilki, and S. Ergün, “Vascular wall-resident stem cells,” Histology and Histopathology, vol. 25, no. 5, pp. 681–689, 2010.
[10]
M. Tavian, B. Zheng, E. Oberlin et al., “The vascular wall as a source of stem cells,” Annals of the New York Academy of Sciences, vol. 1044, pp. 41–50, 2005.
[11]
C. Somasundaram, D. I. Diz, T. Coleman, and R. D. Bukoski, “Adventitial neuronal somata,” Journal of Vascular Research, vol. 43, no. 3, pp. 278–288, 2006.
[12]
B. van der Loo and J. F. Martin, “The adventitia, endothelium and atherosclerosis,” International Journal of Microcirculation-Clinical and Experimental, vol. 17, no. 5, pp. 280–288, 1997.
[13]
S. M. Arribas, C. J. Daly, M. C. González, and J. C. Mcgrath, “Imaging the vascular wall using confocal microscopy,” Journal of Physiology, vol. 584, no. 1, pp. 5–9, 2007.
[14]
W. Warwick, DB: Gray's Anatomy, Churchill Livingstone, Philadelphia, Pa, USA, 37th edition, 1989.
[15]
E. Harno, G. Edwards, A. R. Geraghty et al., “Evidence for the presence of GPRC6A receptors in rat mesenteric arteries,” Cell Calcium, vol. 44, no. 2, pp. 210–219, 2008.
[16]
S. Smajilovic, J. L. Hansen, T. E. H. Christoffersen et al., “Extracellular calcium sensing in rat aortic vascular smooth muscle cells,” Biochemical and Biophysical Research Communications, vol. 348, no. 4, pp. 1215–1223, 2006.
[17]
Y. Wang, E. K. Awumey, P. K. Chatterjee et al., “Molecular cloning and characterization of a rat sensory nerve Ca2+-sensing receptor,” American Journal of Physiology—Cell Physiology, vol. 285, no. 1, pp. C64–C75, 2003.
[18]
R. D. Bukoski, K. Bian, Y. Wang, and M. Mupanomunda, “Perivascular sensory nerve Ca2+ receptor and Ca2+-induced relaxation of isolated arteries,” Hypertension, vol. 30, no. 6, pp. 1431–1439, 1997.
[19]
A. H. Weston, A. Geraghty, I. Egner, and G. Edwards, “The vascular extracellular calcium-sensing receptor: an update,” Acta Physiologica, vol. 203, no. 1, pp. 127–137, 2011.
[20]
M. Ruat, M. E. Molliver, A. M. Snowman, and S. H. Snyder, “Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 8, pp. 3161–3165, 1995.
[21]
S. Mechsner, J. Schwarz, J. Thode, C. Loddenkemper, D. S. Salomon, and A. D. Ebert, “Growth-associated protein 43-positive sensory nerve fibers accompanied by immature vessels are located in or near peritoneal endometriotic lesions,” Fertility and Sterility, vol. 88, no. 3, pp. 581–587, 2007.
[22]
Y. Shen, S. Mani, S. L. Donovan, J. E. Schwob, and K. F. Meiri, “Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system,” Journal of Neuroscience, vol. 22, no. 1, pp. 239–247, 2002.
[23]
Q. He, E. W. Dent, and K. F. Meiri, “Modulation of actin filament behavior by GAP-43 (neuromodulin) is dependent on the phosphorylation status of serine 41, the protein kinase C site,” Journal of Neuroscience, vol. 17, no. 10, pp. 3515–3524, 1997.
[24]
S. M. Goicoechea, D. Arneman, and C. A. Otey, “The role of palladin in actin organization and cell motility,” European Journal of Cell Biology, vol. 87, no. 8-9, pp. 517–525, 2008.
[25]
L. Jin, Q. Gan, B. J. Zieba et al., “The actin associated protein palladin is important for the early smooth muscle cell differentiation,” PLoS ONE, vol. 5, no. 9, Article ID e12823, pp. 1–13, 2010.
[26]
M. J. R？nty, S. K. Leivonen, B. Hinz et al., “Isoform-specific regulation of the actin-organizing protein palladin during TGF-β1-induced myofibroblast differentiation,” Journal of Investigative Dermatology, vol. 126, no. 11, pp. 2387–2396, 2006.
[27]
P. A. Vo and D. R. Tomlinson, “Effects of nerve growth factor on expression of GAP-43 in right atria after sympathectomy in diabetic rats,” Diabetes, Obesity and Metabolism, vol. 3, no. 5, pp. 350–359, 2001.
[28]
S. Y. Tsai, L. Y. Yang, C. H. Wu et al., “Injury-induced Janus kinase/protein kinase C-dependent phosphorylation of growth-associated protein 43 and signal transducer and activator of transcription 3 for neurite growth in dorsal root ganglion,” Journal of Neuroscience Research, vol. 85, no. 2, pp. 321–331, 2007.
[29]
C. C. Toth, D. Willis, J. L. Twiss et al., “Locally synthesized calcitonin gene-related peptide has a critical role in peripheral nerve regeneration,” Journal of Neuropathology and Experimental Neurology, vol. 68, no. 3, pp. 326–337, 2009.
[30]
M. M. Parast and C. A. Otey, “Characterization of palladin, a novel protein localized to stress fibers and cell adhesions,” Journal of Cell Biology, vol. 150, no. 3, pp. 643–655, 2000.
[31]
A. H. Weston, M. Absi, D. T. Ward et al., “Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231,” Circulation Research, vol. 97, no. 4, pp. 391–398, 2005.
