Early and sustained improvements in motor function in rats after infusion of allogeneic umbilical cord-derived mesenchymal stem cells following spinal cord injury.

STUDY DESIGN: Animal study. OBJECTIVES: Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have recently been shown to hold great therapeutic potential for spinal cord injury (SCI). However, majority of the studies have been done using human cells transplanted into the rat with immunosuppression; this may not represent the outcomes that occur in humans. Herein, we present the therapeutic effect of using rat UC-MSCs (rUC-MSC) without immunosuppression in a rat model of SCI. SETTING: Mayo Clinic, Rochester, MN, USA. METHODS: Twelve female rats were randomly divided into two groups, control, and rUC-MSC group, and then subjected to a T9 moderate contusion SCI. Next, 2 × 106 rUC-MSCs or ringer-lactate solution were injected through the tail vein at 7 days post injury. Rats were assessed for 14 weeks by an open-field Basso, Beattie, and Bresnahan (BBB) motor score as well as postmortem quantification of axonal sparing/regeneration, cavity volume, and glial scar. RESULTS: Animals treated with rUC-MSCs were found to have early and sustained motor improvement (BBB score of 14.6 ± 1.9 compared to 10.1 ± 1.7 in the control group) at 14 weeks post injury (mean difference: 4.55, 95% CI: 2.04 to 7.06; p value < 0.001). Total cavity volume in the injury epicenter was significantly reduced in the rUC-MSC group; control: 33.0% ± 2.1, rUC-MSC: 25.3% ± 3.8 (mean difference: -7.7% (95% CI: -12.3 to -2.98); p value < 0.05). In addition, spinal cords from rats treated with rUC-MSCs were found to have a significantly greater number of myelinated axons, decreased astrogliosis, and reduced glial scar formation compared to control rats. CONCLUSIONS: Our study indicates that intravenous injection of allogenic UC-MSCs without immunosuppression exert beneficial effects in subacute SCI and thus could be a useful therapy to improve the functional capacity among patients with SCI.

Overcoming the blood-brain barrier by Annexin A1-binding peptide to target brain tumours.

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.

Rapid changes in shear stress induce dissociation of a G alpha(q/11)-platelet endothelial cell adhesion molecule-1 complex.

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.

The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells.

E-, P- and L-selectins critically function in lymphocyte recirculation and recruiting leukocytes to inflammatory sites. MECA-79 antibody inhibits L-selectin-mediated lymphocyte adhesion in several species and does not require sialic acid in its epitope. Many other antibodies, however, recognize human selectin ligands expressing N-acetylneuraminic acid but not mouse selectin ligands expressing N-glycolylneuraminic acid, suggesting that difference in sialic acid in sialyl Lewis X leads to differential reactivity. We found that HECA-452 and FH6 monoclonal antibodies bind Chinese hamster ovary (CHO) cells expressing N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl form. Moreover, synthetic N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl oligosaccharide inhibited HECA-452 and FH6 binding. By contrast, E-, P- and L-selectin bound to CHO cells regardless of whether they express N-acetyl or N-glycolyl form of sialyl Lewis X, showing that selectins have a broader recognition capacity than HECA-452 and FH-6 anti-sialyl Lewis x antibodies.

Intra-Articular Umbilical Cord Derived Mesenchymal Stem Cell Therapy for Chronic Elbow Osteoarthritis in Dogs: A Double-Blinded, Placebo-Controlled Clinical Trial.

Background: Intra-articular stem cell therapy may help alleviate lameness caused by osteoarthritis in dogs. Umbilical cord-derived stem cell (UMSC) therapy has not yet been investigated in a veterinary clinical study. We hypothesized that dogs treated with intra-articular UMSC will have improved limb function and quality of life when compared to dogs treated with a saline placebo injection. Methods: This was a prospective, double-blinded, placebo-controlled clinical trial in client-owned dogs with chronic elbow osteoarthritis with a follow-up time of 6 months. Dogs were assigned to receive intra-articular UMSC (n = 38) or a saline placebo intra-articular injection (n = 30). Outcome measures included the Canine Brief Pain Inventory score (CBPI) and peak vertical force (PVF) from force-platform gait analysis. Treatment was considered successful when there was a decrease in the Pain Severity Score of at least one and a decrease in the Pain Interference Score of at least one from baseline. Success rates and PVF were compared between groups. Results: No adverse effects associated with UMSC were noted. Of the dogs completing the study, treatment success in the UMSC (n = 28) vs. placebo groups (n = 23) was observed in 54 vs. 28% of dogs at 1 month, 50 vs. 27% at 3 months, and 46 vs. 14% at 6 months, respectively. Success rate in the UMSC group was significantly higher than the placebo group at 1 and 6 months after treatment. However, no differences in PVF of the affected limb over time was observed in either group. Conclusions: Intra-articular UMSC for osteoarthritis may improve clinical signs based on owner observations.

Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.

It is becoming increasingly evident that the cell-cell junction is a major signaling center. Here we show that the Galphaq/11 subunit of heterotrimeric G proteins forms a complex with platelet-endothelial cell adhesion molecule 1 (PECAM-1), a junctional protein that has been shown to be involved in mechanosignaling in endothelial cells. To understand the role of PECAM-1 in this complex, we determined the critical regions of PECAM-1 involved in this interaction. By expressing truncated forms of PECAM-1 in human embryonic kidney (HEK293) cells, we found that the cytoplasmic domain of PECAM-1 is not required for its association with Galphaq/11. Domain swapping of PECAM-1 with intracellular cell adhesion molecule 1 (ICAM-1), a protein that does not form a complex with Galphaq/11, provides evidence that the extracellular domain of PECAM-1 is critical for this interaction. This result also suggests that PECAM-1 does not directly interact with Galphaq/11. Coexpression of bradykinin receptor B2 (BKRB2), a Galphaq/11-coupled receptor, with PECAM-1 enhances formation of the PECAM-1-Galphaq/11 complex, suggesting an interaction between PECAM-1 and BKRB2. Co-immunoprecipitation experiments indicate that these two molecules indeed form a complex when expressed in HEK293 cells. Activation of ERK1/2 by bradykinin in HUVEC is enhanced when PECAM-1 expression is inhibited by transfection of small interference RNA against PECAM-1. Taken together, our results provide evidence of interaction of PECAM-1 with BKRB2 and of its possible role in regulating G protein-coupled receptor (GPCR) and G protein functions.

PECAM-1 mediates NO-dependent dilation of arterioles to high temporal gradients of shear stress.

OBJECTIVE: In response to changes in wall shear stress (WSS) the vascular endothelium releases several factors, among others nitric oxide. On the basis of studies of endothelial cells in culture, suggesting that platelet endothelial cell adhesion molecule-1 (PECAM-1) is specifically involved in sensing and coupling high temporal gradients of fluid shear stress with activation of eNOS, we hypothesized that dilations of isolated skeletal muscle arterioles from PECAM-1 knockout mice (PECAM-KO) will be reduced to rapid increases in WSS elicited by increases in perfusate flow. METHODS AND RESULTS: Small and large step increases in flow resulted in substantial dilations in arterioles of WT mice (45+/-4%), but they were markedly reduced in arterioles of PECAM-KO mice (22+/-5%). The initial slope of dilations, when WSS increased rapidly, was greater in vessels of WT than those of PECAM-KO mice (slopes: 0.378 and 0.094, respectively), whereas the second phase of dilations, when flow/shear stress was steady, was similar in the 2 groups (slopes: 0.085 and 0.094, respectively). Inhibition of eNOS significantly reduced the initial phase of dilations in arterioles from WT, but not from those of PECAM-KO mice. The calcium ionophore A23187 elicited similar NO-mediated dilation in both WT and PECAM-KO mice. CONCLUSIONS: In isolated arterioles of PECAM-KO mice activation of eNOS and consequent dilation by agonists is maintained, but the dilation to high temporal gradients of wall shear stress elicited by increases in perfusate flow is reduced. Thus, we propose that PECAM-1 plays an important role in the ability of the endothelium to sense and couple high temporal gradients of wall shear stress to NO-mediated arteriolar dilation during sudden changes in blood flow in vivo.

Skeletal adaptation to intramedullary pressure-induced interstitial fluid flow is enhanced in mice subjected to targeted osteocyte ablation.

Interstitial fluid flow (IFF) is a potent regulatory signal in bone. During mechanical loading, IFF is generated through two distinct mechanisms that result in spatially distinct flow profiles: poroelastic interactions within the lacunar-canalicular system, and intramedullary pressurization. While the former generates IFF primarily within the lacunar-canalicular network, the latter generates significant flow at the endosteal surface as well as within the tissue. This gives rise to the intriguing possibility that loading-induced IFF may differentially activate osteocytes or surface-residing cells depending on the generating mechanism, and that sensation of IFF generated via intramedullary pressurization may be mediated by a non-osteocytic bone cell population. To begin to explore this possibility, we used the Dmp1-HBEGF inducible osteocyte ablation mouse model and a microfluidic system for modulating intramedullary pressure (ImP) to assess whether structural adaptation to ImP-driven IFF is altered by partial osteocyte depletion. Canalicular convective velocities during pressurization were estimated through the use of fluorescence recovery after photobleaching and computational modeling. Following osteocyte ablation, transgenic mice exhibited severe losses in bone structure and altered responses to hindlimb suspension in a compartment-specific manner. In pressure-loaded limbs, transgenic mice displayed similar or significantly enhanced structural adaptation to Imp-driven IFF, particularly in the trabecular compartment, despite up to ∼50% of trabecular lacunae being uninhabited following ablation. Interestingly, regression analysis revealed relative gains in bone structure in pressure-loaded limbs were correlated with reductions in bone structure in unpressurized control limbs, suggesting that adaptation to ImP-driven IFF was potentiated by increases in osteoclastic activity and/or reductions in osteoblastic activity incurred independently of pressure loading. Collectively, these studies indicate that structural adaptation to ImP-driven IFF can proceed unimpeded following a significant depletion in osteocytes, consistent with the potential existence of a non-osteocytic bone cell population that senses ImP-driven IFF independently and potentially parallel to osteocytic sensation of poroelasticity-derived IFF.

Type II cGMP-dependent protein kinase mediates osteoblast mechanotransduction.

Continuous bone remodeling in response to mechanical loading is critical for skeletal integrity, and interstitial fluid flow is an important stimulus for osteoblast/osteocyte growth and differentiation. However, the biochemical signals mediating osteoblast anabolic responses to mechanical stimulation are incompletely understood. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/DeltafosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity. Fluid shear stress increased osteoblast nitric oxide (NO) synthesis, leading to activation of cGMP-dependent protein kinase (PKG). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membrane-permeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression. In cells transfected with small interfering RNAs (siRNA) specific for membrane-bound PKG II, shear- and cGMP-induced Erk activation and fos family gene expression was nearly abolished and could be restored by transducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. Thus, we report a novel function for PKG II in osteoblast mechanotransduction, and we propose a model whereby NO/cGMP/PKG II-mediated Erk activation and induction of c-fos, fra-1, fra-2, and fosB/DeltafosB play a key role in the osteoblast anabolic response to mechanical stimulation.

PECAM-1 is a critical mediator of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of large arteries in which lesion development preferentially occurs at vessel sites exposed to rapid changes in flow. We have previously shown that platelet endothelial cell adhesion molecule (PECAM-1), a surface receptor of the immunoglobulin superfamily, is involved in mechanosensing of rapid changes in flow. We wondered whether apolipoprotein E deficient (ApoE(-/-)) mice, predisposed to development of atheromas, would be protected from atherosclerosis by deficiency in PECAM-1. Using double knockout (DKO) mice for both PECAM-1 and ApoE (ApoE(-/-)/PECAM-1(-/-)) we found a significant reduction of sudanophilic lesions in their aortae compared to single knockout (SKO) (ApoE(-/-)/PECAM-1(+/+)) mice maintained on a high-fat Western diet. Immunostaining of aortic sinus cross sections demonstrated significantly lower ICAM-1 expression in DKO lesions compared with SKO lesions, and en face preparations of vessel regions subjected to disturbed and laminar flow showed less disruption of junctional connexin 43 in DKO than in SKO mice. Thus, PECAM-1 deficiency reduced the extent of lesions at the aortic arch and the aortic sinus, and lowered atherogenic indices. These results suggest that PECAM-1 is an important factor in the atherogenic changes seen in the ApoE-deficient mouse model and thus should be considered as a potential target for protection against atherosclerosis.

Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis X-type L-selectin ligand activity.

It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E- and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAcalpha2-->3Galbeta1-->4(Fucalpha1-->3(sulfo-->6))GlcNAcbeta1--> 3Galbeta1-->3GalNAcalpha1-->Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that beta1,3-N-acetylglucosaminyltransferase-3 (beta3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different beta3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding beta3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 beta1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either beta3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.

Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM.

The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.