Given the importance of the cartilage endplate (CEP) in low back pain (LBP), there is a need to characterize the human CEP at the molecular, cell, and tissue levels to inform treatment strategies that target it. The goal of this study was to characterize the structure, matrix composition, and cell phenotype of the human CEP compared with adjacent tissues within the intervertebral joint: the nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC). Isolated CEP, NP, AF, and AC tissues and cells were evaluated for cell morphology, matrix composition, collagen structure, glycosaminoglycan content, and gene and protein expression. The CEP contained elongated cells that mainly produce a collagen-rich interterritorial matrix and a proteoglycan-rich territorial matrix. The CEP contained significantly fewer glycosaminoglycans than the NP tissue. Significant differences in matrix and cell marker gene expression were observed between CEP and NP or AF, with the greatest differences between CEP and AC. We were able to distinguish NP from CEP cells using collagen-10 (COLX), highlighting COLX as a potential CEP marker.Our findings suggest that at the cell and tissue levels, the CEP demonstrates both similarities and differences when compared with NP, AF, and hyaline AC. This study highlights a unique structure, matrix composition, and cell phenotype for the human CEP and can help to inform regenerative strategies that target the intervertebral disc joint in chronic LBP. K E Y W O R D S cartilage endplate, cell phenotype, characterization, intervertebral disc, low back pain 1 | INTRODUCTION Low back pain (LBP) is a major source of socioeconomic burden worldwide with an annual cost of greater than $100 billion in the United States. 1 It is the second most frequent reason for visits to primary care physicians 1 and opioid prescription. 2 Despite a lifetime prevalence of LBP at 84%, 3 the underlying mechanisms of LBP remain unknown. Nonspecific LBP is highly associated with lumbar intervertebral disc (IVD) degeneration, 3 but treatment strategies are limited and fail to target the underlying cellular mechanisms implicated in this disease.The healthy IVD is avascular and aneural and provides pain-free movement of the spinal column. It is composed of a gelatinous core of proteoglycans, the nucleus pulposus (NP), concentric rings of type-1 collagen, the annulus fibrosus (AF), and presumably hyaline-like cartilage
Abdominal aortic aneurysm (AAA) is a localized pathological dilation of the aorta exceeding the normal diameter (∼20 mm) by more than 50% of its original size (≥30 mm), accounting for approximately 150000–200000 deaths worldwide per year. We previously reported that Notch inhibition does not decrease the size of pre-established AAA at late stage of the disease. Here, we examined whether a potent pharmacologic inhibitor of Notch signaling (DAPT (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester)), regresses an actively growing AAA. In a mouse model of an aneurysm (Apoe−/− mice; n=44); DAPT (n=17) or vehicle (n=17) was randomly administered at day 14 of angiotensin II (AngII; 1 µg/min/kg), three times a week and mice were killed on day 42. Progressive increase in aortic stiffness and maximal intraluminal diameter (MILD) was observed in the AngII + vehicle group, which was significantly prevented by DAPT (P<0.01). The regression of aneurysm with DAPT was associated with reduced F4/80+Cd68+ (cluster of differentiation 68) inflammatory macrophages. DAPT improved structural integrity of aorta by reducing collagen fibrils abnormality and restoring their diameter. Mechanistically, C–C chemokine receptor type 7 (Ccr7)+F4/80− dendritic cells (DCs), implicated in the regression of aneurysm, were increased in the aorta of DAPT-treated mice. In the macrophages stimulated with AngII or lipopolysaccharide (LPS), DAPT reverted the expression of pro-inflammatory genes Il6 and Il12 back to baseline within 6 h compared with vehicle (P<0.05). DAPT also significantly increased the expression of anti-inflammatory genes, including c-Myc, Egr2, and Arg1 at 12–24 h in the LPS-stimulated macrophages (P<0.05). Overall, these regressive effects of Notch signaling inhibitor emphasize its therapeutic implications to prevent the progression of active AAAs.
Background: Platelet adhesion to the subendothelial collagen fibrils is one of the first steps in hemostasis. Understanding how structural perturbations in the collagen fibril affect platelet adhesion can provide novel insights into disruption of hemostasis in various diseases. We have recently identified the presence of abnormal collagen fibrils with compromised D-periodic banding in the extracellular matrix remodeling present in abdominal aortic aneurysms (AAA).Objective: In this study, we employed multimodal microscopy approaches to characterize how collagen fibril structure impacts platelet adhesion in clinical AAA tissues.Methods: Ultrastructural atomic force microscopy (AFM) analysis was performed on tissue sections after staining with fluorescently labeled collagen hybridizing peptide (CHP) to recognize degraded collagen. Second harmonic generation (SHG) microscopy was used on CHP-stained sections to identify regions of intact versus degraded collagen. Finally, platelet adhesion was identified via SHG and indirect immunofluorescence on the same tissue sections. Results:Our results indicate that ultrastructural features characterizing collagen fibril abnormalities coincide with CHP staining. SHG signal was absent from CHP-positive regions. Additionally, platelet binding was primarily localized to regions with SHG signal. Abnormal collagen fibrils present in AAA (in SHG negative regions) were thus found to inhibit platelet adhesion compared to normal fibrils. Conclusions:Our investigations reveal how the collagen fibril structure in the vessel wall can serve as another regulator of platelet-collagen adhesion. These results can be broadly applied to understand the role of collagen fibril structure in regulating thrombosis or bleeding disorders.
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