ADAM8 is selectively up-regulated in endothelial cells and is associated with angiogenesis after spinal cord injury in adult mice

Abstract

Endothelial cell (EC) loss and subsequent angiogenesis occur over the first week after spinal cord injury (SCI). To identify molecular mechanisms that could be targeted with intravenous (i.v.) treatments, we determined whether transmembrane "a disintegrin and metalloprotease" (ADAM) proteins are expressed in ECs of the injured spinal cord. ADAMs bind to integrins, which are important for EC survival and angiogenesis. Female adult C57Bl/6 mice with a spinal cord contusion had progressively more ADAM8 (CD156) immunostaining in blood vessels and individual ECs between 1 and 28 days following injury. Uninjured spinal cords had little ADAM8 staining. The increase in ADAM8 mRNA and protein was confirmed in spinal cord lysates, and ADAM8 mRNA was present in FACS-enriched ECs. ADAM8 colocalized extensively and exclusively with the EC marker PECAM and also with i.v.-injected lectins. Intravenous isolectin B4 (IB4) labels a subpopulation of blood vessels at and within the injury epicenter 3-7 days after injury, coincident with angiogenesis. Both ADAM8 and the proliferation marker Ki-67 were present in IB4-positive microvessels. ADAM8-positive proliferating cells were seen at the leading end of IB4-positive blood vessels. Angiogenesis was confirmed by BrdU incorporation, binding of i.v.-injected nucleolin antibodies, and MT1-MMP immunostaining in a subset of blood vessels. These data suggest that ADAM8 is vascular selective and plays a role in proliferation and/or migration of ECs during angiogenesis following SCI.

Figure 1. ADAM8 immunostaining increases following SCI in adult mice

A-F) Shown are horizontal (longitudinal) sections through the center of the spinal cords of uninjured mice (0) and at progressively increasing times after injury. Left is rostral. In uninjured mice (A) very little ADAM8 immunostaining is seen. Positive structures appeared to be blood vessels as shown in the inset. At 1 and 3 days post-injury (B,C), some ADAM8-positive structures can be seen around the epicenter (*), which itself appears devoid of staining. From 7 days onward, progressively more staining appears, particularly in the epicenter (D-F), the site where lost tissue is replaced by a fibroblast- and laminin-rich cell matrix. At all post-injury times ADAM8-positive blood vessels could be seen often times in the penumbra (insets). G) The area occupied by ADAM8-positive structures in horizontal sections throughout the dorso-ventral extent of the spinal cord shows that the increase becomes significantly different from the uninjured controls by 7 days post-injury. Data are means plus standard deviation; n = 4 each.

Figure 2. ADAM8 mRNA and protein is increased following SCI

A) Western blots show that of whole spinal cord extract from uninjured (0) and 7 day injured (7d SCI) mice probed with ADAM8 antibody shows two bands around the expected 80 kDa size of mature ADAM8. The doublet probably represents different post-translationally modified forms. The lack of other bands shows the specificity of the antibody. A blot of trachea extract is shown as a positive control. Note the increase of ADAM8 7 days after injury compared to uninjured control, which was seen in 3 different experiments. B) End-point RT-PCR shows the absence of ADAM8 mRNA in 2 uninjured spinal cord extracts (0) and the increased expression in individual mice at both 1 and 3 days post-injury. The bands are at 459 bp, the expected amplicon size for the primer set used. For the negative control, reverse transcriptase was replaced by water. C) RT-PCR of whole spinal cord at 3 days post-injury (3d SCI whole; 2 lanes = 2 mice) was compared to that of FACS-purified spinal microvascular ECs (smvECs) pooled from 12 mice per lane 3 days following SCI. The household gene cyclophilin A (cA) is shown for comparison. Despite the much reduced mRNA levels in the isolated preparation, judged by the reduced cA mRNA, the ADAM8 mRNA remains detectable, suggesting an enrichment of ADAM8 in blood vessels. Solid arrowhead indicates the expected amplicon size of ADAM8 and the open arrowhead the expected amplicon size of cA.

Figure 3. ADAM8 is present in perfused blood vessels and within ECs of the injury epicenter following SCI

Shown is a horizontal section through the center of the spinal cord at 7 days post-injury. The lateral side is on the right, the rostral side at the top and the central canal would be ∼1/3 from the left. ADAM8 shows extensive co-localization with PECAM, a luminal marker for ECs. ADAM8 is seen not only in penumbral blood vessels (arrows) around the injury epicenter (*), many of which are labeled by i.v. injection of LEA, but also within EC structures within the epicenter, which typically is devoid of perfused blood vessels. Arrows indicate clear examples of perfused blood vessels that also have ADAM8 and PECAM staining.

Figure 4. ADAM8 is present in angiogenic blood vessels following SCI

A-D) Seven days post-injury, ADAM8 (A) is seen within several blood vessels overlapping with PECAM (B) and i.v. injected IB4 lectin (C) both of which are known to be present on the luminal side of ECs. The arrows in the merged confocal image (D) indicate the abluminal rim of ADAM8 staining, suggesting the interaction with the region between the ECs and the basement membrane. The circular structures on the bottom left are probably cross sections through three blood vessel branches. Note also that more structures are positive for ADAM8 and PECAM than for IB4 suggesting that these are non-perfused blood vessels. E-H) Seven days following injury, ADAM8 immunostaining (E) can be seen associated with a blood vessel that was labeled by i.v. injection of IB4 (F). Ki-67-positive (G), proliferating, ECs (arrows) are seen in the thinner IB4-positive branch as well as in an ADAM8-positive cell emanating from the leading non-perfused end (H).

Figure 5. Angiogenic markers are present following SCI

A-C) To label proliferating cells, BrdU was injected systemically over the last 24 hours up to 3 days post-injury. BrdU-positive nuclei can be seen throughout a small region of the spinal cord. Some of the BrdU-positive nuclei co-localize with PECAM-positive ECs (B) as is evident in the merged image (C). D-F) Some perfused blood vessels, identified by i.v. injected LEA (D), can also be labeled by i.v. injection of antibodies against nucleolin (E), a marker for angiogenic blood vessels in tumors, as is evident from the merged confocal image (F). G-I) Some tips of PECAM-positive blood vessels (G) contain MT1-MMP (arrows in H), a marker for migrating ECs, as shown in the merged confocal image (I).