Expression of PTHrP and its cognate receptor in the rheumatoid synovial microcirculation

doi:10.1016/S0006-291X(02)02263-5

Biochemical and Biophysical Research Communications

Volume 297, Issue 4, 4 October 2002, Pages 890-897

https://doi.org/10.1016/S0006-291X(02)02263-5 Get rights and content

Abstract

Parathyroid hormone-related protein (PTHrP), a multifunctional peptide that acts as a vasodilator as well as possible regulator of vascular development, is produced in increased amounts in the rheumatoid synovium. To understand whether PTHrP can contribute to the development and function of the rheumatoid microcirculation, studies were undertaken to identify and compare vascular sites of expression of PTHrP and its cognate receptor in the rheumatoid synovium and/or in cultured rheumatoid synovial endothelial cells. Endothelial cells, including apoptotic cells, as determined by TUNEL staining, were the primary site of vascular PTHrP expression in the rheumatoid synovium, a result confirmed in vitro in rheumatoid synovial microvascular endothelial cells. In contrast, the PTH/PTHrP receptor was primarily located in pericytes and smooth muscle cells within the vasculature. These results are consistent with a possible paracrine pathway for PTHrP action in the synovial microcirculation, wherein PTHrP peptides secreted by the synovial endothelium could act on surrounding PTH1R-positive pericytes and smooth muscle cells.

Section snippets

Materials and methods

Immunohistochemical analysis of rheumatoid synovial tissue. Synovial tissue specimens obtained from patients with rheumatoid arthritis at the time of joint replacement surgery (n=5) were immediately fixed in 10% formalin and embedded in paraffin for immunohistochemical staining. Using previously described methods [5], tissues were processed for detection of PTHrP or the PTH/PTHrP receptor (PTH1R) using affinity purified rabbit polyclonal antibodies generated against either human PTHrP(34–53)

Expression of PTHrP in the rheumatoid synovial microcirculation

Endothelial cells (Fig. 1A) were the primary cellular site of vascular PTHrP expression in human rheumatoid synovial tissue (Table 1), as determined by immunohistochemical staining (Figs. 1B, E, G, and H). In all cases, specificity of PTHrP immunoreactivity was verified by the absence of staining that resulted on sequential sections treated with PTHrP antibody that had been preincubated with an excess of antigen. PTHrP-positive endothelial cells were present in all of the rheumatoid synovial

Discussion

This study provides a unique opportunity to examine the expression of PTHrP and its cognate receptor in the microcirculation of a human tissue that is undergoing active vascular remodeling. In the rheumatoid synovium, immunohistochemical studies identified endothelial cells as the primary site of vascular PTHrP expression, a finding further supported by the demonstration of PTHrP mRNA expression and protein secretion from cultured microvascular endothelial cells isolated from the rheumatoid

Acknowledgements

This work was supported by a grant from the Arthritis Foundation to J.L. Funk.

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Rian and collaborators (Rian et al., 1994) showed for the first time that PTHrP, but not the PTH/PTHrP receptor, is produced by human endothelial cells. As smooth muscle cells express PTH/PTHrP receptors (Funk et al., 2002), these investigators suggest that the hormone produced by endothelial cells may has a paracrine action on these muscle cells (Diamond et al., 2006), however, they cannot rule out an autocrine effect of PTHrP. Curiously, there are controversial results about the effects of PTHrP on angiogenesis.
We showed that Parathyroid Hormone-related Peptide (PTHrP) induces proliferation, migration, survival and chemoresistance via MAPKs and PI3K/AKT pathways in colorectal cancer (CRC) cells. The objective of this study was to investigate if PTHrP is also involved in tumor angiogenesis. PTHrP increased VEGF expression and the number of structures with characteristics of neoformed vessels in xenografts tumor. Also, PTHrP increased mRNA levels of VEGF, HIF-1α and MMP-9 via ERK1/2 and PI3K/Akt pathways in Caco-2 and HCT116 cells. Tumor conditioned media (TCMs) from both cell lines treated with PTHrP increases the number of cells, the migration and the tube formation in the endothelial HMEC-1 cells, whereas the neutralizing antibody against VEGF diminished this response. In contrast, PTHrP by direct treatment only increased ERK1/2 phosphorylation and the HMEC-1 cells number. These results provide the first evidence related to the mode of action of PTHrP that leads to its proangiogenic effects in the CRC.
Effects of PTH [1-34] on synoviopathy in an experimental model of osteoarthritis preceded by osteoporosis
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Synoviopathy contributes to cartilage degradation in osteoarthritis (OA). Intermittent parathyroid hormone (PTH) [1-34] administration inhibits terminal differentiation of human chondrocytes and prevents cartilage damage. We aimed to determine whether PTH [1-34] could modify synovial changes in experimental OA preceded by osteoporosis (OP).
Twenty osteoporosis (OP) rabbits underwent knee surgery to induce OA. They were administered either saline vehicle or PTH for 10 weeks. Ten healthy rabbits were used as controls. Following sacrifice, synovial changes were assessed by Krenn synovitis score, immunohistochemistry for macrophages (RAM-11), B and T lymphocytes, type I collagen, parathyroid hormone 1 receptor (PTH1R), and anti-proliferating cell nuclear antigen (PCNA). Synovial mRNA levels of Col1A1, IL-1β, cyclooxygenase 2 (COX-2), matrix-degrading metalloproteinases (MMP-9, MMP-13), and monocyte chemotactic protein-1 (MCP-1), as well as protein expression of PTH1R were also determined. Cartilage damage was analyzed by Mankin score.
OPOA + vehicle rabbits showed an increase in synovitis score vs controls (P = 0.003), mainly due to synovial hyperplasia and fibrosis, while PTH reduced these changes (P = 0.017). Mankin and Krenn scores were well correlated in all groups (r = 0.629, P = 0.012). Immunostaining for RAM-11 and B lymphocytes was increased (P ≤ 0.05), whereas PTH1R protein levels tended to be higher in OPOA + vehicle animals vs controls. PTH did not modify RAM-11 staining or PTH1R levels; however, it restored PTH1R localization to the vicinity of synovial vessels. PTH also decreased type I collagen, MCP-1, and MMP-13 expression (P < 0.05), as well as PCNA staining compared to vehicle-treated OPOA rabbits.
In our model of OA aggravated by previous OP, synoviopathy correlated well with cartilage damage. Intermittent PTH [1-34] administration ameliorated both hyperplasia and fibrosis.
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The inflammatory response in pancreatitis is accompanied by an increase in cytokine and chemokine levels. PTHrP exerts pro-inflammatory effects in the injured kidney, atherosclerosis and rheumatoid arthritis [13–17]. Here we measured the effects of PTHrP on IL-6 and ICAM-1 mRNA levels.
Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1–36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1–36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7–34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.
Induction of cardiovascular pathology in a novel model of low-grade chronic inflammation
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However, caution must be urged in universally applying this association because in certain instances such as burn injury [38] and pancreatitis [39], hypocalcemia occurs in the absence of a hyperparathyroid response. Up-regulation of hepatic PTHrP expression has been reported in association with the acute phase response that accompanies sepsis [40], in central nervous system inflammation resulting to brain injury [41], and in the synovial joints of patients with rheumatoid arthritis [42]. Increased PTHrP and SHPT are especially relevant to the osteopenic response reported previously in this model of chronic inflammation [13], and future studies should consider their role in bone and vascular pathology.
Epidemiological and clinical evidence indicate that inflammatory processes play a pivotal role in a number of conditions associated with aging, including osteoporosis and cardiovascular diseases. The purpose of this study was to evaluate cardiovascular pathology and select inflammatory mediators of interest in a model of low-grade inflammation-induced osteopenia.
Slow-release pellets were subcutaneously implanted in male rats to deliver 0, 3.3, or 33.3 μg of lipopolysaccharide (LPS)/day for 90 days. Tail blood was collected at 1, 2, and 3 months for differential white cell counts, and at the end of the study, hearts were harvested for histological and immunohistochemical evaluation.
The low-grade inflammatory response was characterized by elevated peripheral blood neutrophils and monocytes. Histological examination of heart cross sections revealed increased fibrous tissue, infiltration of lymphocytes, accumulation of mast cells, and roughened intimal borders within the arteries and arterioles, consistent with vascular disease. Inflammatory mediators (cyclooxygenase-2, tumor necrosis factor-α, and interleukin-1β) were up-regulated, and increased expression of platelet endothelial cell adhesion molecule-1 and receptor activator for NF-κB ligand was localized to the microvasculature endothelium.
These findings suggest that inflammation induced by chronic exposure to LPS produces cardiovascular pathology in the smaller intramural arteries and arterioles and support the utility of this model for further mechanistic and therapeutic studies focused on the role of chronic inflammation in cardiovascular disease.
The fibroblast-like synovial cell in rheumatoid arthritis: A key player in inflammation and joint destruction
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Although multiple cell types are present in the rheumatoid joint, the fibroblast-like synovial cell (FLS) is among the most prominent. It is now appreciated that the FLS is not only space-filling, but is directly responsible for cartilage destruction, and also drives both inflammation and autoimmunity. In this article, we consider the normal role of the FLS in healthy joints, and review evidence that implicates the FLS as a central player in the propagation of rheumatoid arthritis.
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¹: Present address. Pfizer Global Research & Development, San Diego, USA.

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Expression of PTHrP and its cognate receptor in the rheumatoid synovial microcirculation

Abstract

Section snippets

Materials and methods

Expression of PTHrP in the rheumatoid synovial microcirculation

Discussion

Acknowledgements

Am. J. Obstet. Gynecol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Am. J. Obstet. Gynecol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Brain Res.

Bone

Blood

Starving the synovium: angiogenesis and inflammation in RA

J. Clin. Invest.

Focally regulated endothelial proliferation and cell death in human synovium

Am. J. Path

Matrix metalloproteinase 9, apoptosis, and vascular morphology in early arthritis

Arthritis Rheum.

Synovial lining, endothelial and inflammatory mononuclear cell proliferation in synovial membranes in psoriatic and reactive arthritis

Br. J. Rheum.

Synovium as a source of increased amino-terminal PTHrP expression in RA

J. Clin. Invest.

The increase of PTHrP and cytokine levels in synovial fluid of elderly RA and OA

Endocr. J.

PTHrP in synovial fluid and disease activity of RA

Br. J. Rheumatol.

Synovial fluids from patients with OA and RA contain high levels of PTHrP

J. Bone Miner. Res.