Roeland Hanemaaijer

Cathepsin K Is the Principal Protease in Giant Cell Tumor of Bone

Giant cell tumor (GCT) of bone is a neoplasm of bone characterized by a localized osteolytic lesion. The nature of GCT is an enigma and the cell type(s) and protease(s) responsible for the extensive localized clinicoradiological osteolysis remain unresolved. We evaluated protease expression and cellular distribution of the proteolytic machinery responsible for the osteolysis. mRNA profiles showed that cathepsin K, cathepsin L, and matrix metalloproteinase (MMP)-9 were the preferentially expressed collagenases. Moderate expression was found for MMP-13, MMP-14, and cathepsin S. Specific protease activity assays revealed high cathepsin K activity but showed that MMP-9 was primarily present (98%) as inactive proenzyme. Activities of MMP-13 and MMP-14 were low. Immunohistochemistry revealed a clear spatial distribution: cathepsin K, its associated proton pump V-H(+)-ATPase, and MMP-9 were exclusively expressed in osteoclast-like giant cells, whereas cathepsin L expression was confined to mononuclear cells. To explore a possible role of cathepsin L in osteolysis, GCT-derived, cathepsin L-expressing, mononuclear cells were cultured on dentine disks. No evidence of osteolysis by these cells was found. These results implicate cathepsin K as the principal protease in GCT and suggest that osteoclast-like giant cells are responsible for the osteolysis. Inhibition of cathepsin K or its associated proton-pump may provide new therapeutic opportunities for GCT.

The pathophysiology of abdominal aortic aneurysm growth: corresponding and discordant inflammatory and proteolytic processes in abdominal aortic and popliteal artery aneurysms.

OBJECTIVEnThere is remarkable controversy over the processes driving abdominal aneurysm growth. The inherent limitations of animal and human studies hamper elucidation of the key inflammatory and proteolytic processes. Human data are largely derived from surgical specimens that typically reflect the final stages of the disease process and thus do not allow distinction between primary and secondary processes. Clear epidemiologic and genetic associations between abdominal aortic aneurysm (AAA) and popliteal artery aneurysms (PAA) suggest that that these two pathologies share common grounds. On this basis, we reasoned that information of corresponding and discordant processes in these aneurysms might provide critical clues on the processes that are crucial for aneurysm progression.nnnMETHODSnMessenger RNA (semi-quantitative real-time polymerase chain reaction) and protein analysis (enzyme-linked immunosorbent assay, multiplex, Western blotting), and histology were performed on aneurysm wall samples obtained during elective PAA and AAA repair. Nonaneurysmal aorta tissue from organ donors was included as reference.nnnRESULTSnMessenger RNA and protein analysis showed that PAA and AAA are both characterized by a marked activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) proinflammatory transcription factors, and hyperexpression of interleukin (IL)-6 and IL-8. Discordant findings were found for other inflammatory markers such as interferon-gamma, interferon-inducible protein 10, tumor necrosis factor-alpha, monocyte chemotactic protein-1, and macrophage inflammatory protein 1alpha and beta, which were all lower in PAA. On the cellular level, both pathologies exhibited profuse infiltration of macrophages, neutrophils, and T-helper cells. Results for B cells, plasma cells, and cytotoxic T cells were discordant, with minimal infiltration of these cell types in PAA. Evaluation of protease expression and activation showed that both conditions are dominated by increased matrix metalloproteinase 8 and 9, and cathepsin K, L and S expression and activation.nnnCONCLUSIONnThis explorative study characterizes degenerative aneurysmal disease general inflammatory conditions that are dominated by profound activation of the NF-kappaB and AP-1 pathways, hyperexpression of IL-6 and IL-8, and neutrophil involvement. Discordant findings for interferon gamma, cytotoxic T cells, B cells, and plasma cells challenge a critical role for these factors in the process of aneurysm growth. Pharmaceutic strategies targeting the common components in AAA and PAA may prove effective for the stabilization of AAA.

Doxycycline therapy for abdominal aneurysm: Improved proteolytic balance through reduced neutrophil content

BACKGROUNDnMatrix metalloproteinase-9 (MMP-9) is thought to play a central role in abdominal aortic aneurysm (AAA) initiation. Doxycycline, a tetracycline analogue, has direct MMP-9-inhibiting properties in vitro, and it effectively suppresses AAA development in rodents. Observed inhibition of AAA progression, and contradictory findings in human studies evaluating the effect of doxycycline therapy on aortic wall MMP-9, suggest that the effects of doxycycline extend beyond MMP-9 inhibition and that the effect may be dose-dependent.nnnMETHODSnThis clinical trial evaluated the effect of 2 weeks of low- (50 mg/d), medium- (100 mg/d), or high-dose (300 mg/d) doxycycline vs no medication in four groups of 15 patients undergoing elective AAA repair. The effect of doxycycline treatment on MMP and cysteine proteases, and their respective inhibitors, was evaluated by quantitative polymerase chain reaction, Western blot analysis, immunocapture protease activity assays, and immunohistochemistry.nnnRESULTSnDoxycycline was well tolerated and no participants dropped out. Doxycycline treatment reduced aortic wall MMP-3 and MMP-25 messenger RNA expression (P < .045 and P < .014, respectively), selectively suppressed neutrophil collagenase and gelatinase (MMP-8 and MMP-9) protein levels (P < .013 and <.004, respectively), and increased protein levels of the protease inhibitors tissue inhibitor of metalloproteinase 1 and cystatin C (P < .029). As for the apparent selective effect on neutrophil-associated proteases, we sought for a reducing effect on aortic wall neutrophil content that was indeed confirmed by immunohistochemical analysis that revealed a 75% reduction in aneurysm wall neutrophil content (P < .001).nnnCONCLUSIONSnIndependent of its dose, short-term preoperative doxycycline therapy improves the proteolytic balance in AAA, presumably through an effect on aortic wall neutrophil content. This study provides a rationale for doxycycline treatment in patients with an AAA as well as in other (vascular) conditions involving neutrophil influx such as Kawasaki disease and Behçet disease.

A Clinical Evaluation of Statin Pleiotropy: Statins Selectively and Dose-Dependently Reduce Vascular Inflammation

Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFκB regulated mediators (i.e. IL-6 (P<0.001) and MCP-1 (P<0.001)); shifted macrophage polarization towards a M2 phenotype (P<0.0003); selectively reduced macrophage-related markers such as cathepsin K and S (P<0.009 and 0.0027 respectively), and ALOX5 (P<0.0009), and reduced vascular wall NFκB activity (40 mg/day group, P<0.016). No effect was found on other cell types. Evaluation of the clinical efficacy of statins to reduce AAA progression did not indicate an effect of statins on aneurysm growth (P<0.337). Hence, in the context of AAA the clinical relevance of statins pleiotropy appears minimal.

Pathogenic Sequence for Dissecting Aneurysm Formation in a Hypomorphic Polycystic Kidney Disease 1 Mouse Model

Objective— Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1nl/nL mouse model with dissecting aneurysms in the aorta. Methods and Results— Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding. Conclusions— The Pkd1nl/nl mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies.

Impaired Collagen Biosynthesis and Cross-linking in Aorta of Patients With Bicuspid Aortic Valve

Background Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm. In the present study, collagen homeostasis in nondilated and dilated aorta segments from patients with BAV was studied, with normal and dilated aortas from tricuspid aortic valve (TAV) patients as reference. Methods and Results Ascending aortas from 56 patients were used for biochemical and morphological analyses of collagen. mRNA expression was analyzed in 109 patients. Collagen turnover rates were similar in nondilated and dilated aortas of BAV patients, showing that aneurysmal formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. However, BAV in general was associated with an increased aortic collagen turnover compared with nondilated aortas of TAV patients. Importantly, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), 2 distinct forms of collagen cross‐linking, was lower in dilated aortas from patients with BAV, which suggests that BAV is associated with a defect in the posttranslational collagen modification. This suggests a deficiency at the level of lysyl hydroxylase (PLOD1), which was confirmed by mRNA and protein analyses that showed reduced PLOD1 expression but normal lysyl oxidase expression in dilated aortas from patients with BAV. This suggests that impaired collagen cross‐linking in BAV patients may be attributed to changes in the expression and/or activity of PLOD1. Conclusions Our results demonstrate an impaired biosynthesis and posttranslational modification of collagen in aortas of patients with BAV, which may explain the increased aortic aneurysm formation in BAV patients.

Inhibition of matrix metalloproteinases (MMPs) by tetracyclines

Remodelling of the extracellular matrix and destruction of connective tissue are regarded as characteristic features of degenerative and invasive processes such as rheumatoid arthritis, periodontitis, wound healing, tumour growth and metastatic invasion. Matrix remodelling is a complex process in which matrix metalloproteinases (MMPs) play a central role. MMP activity arises from a multi-step process, which is tightly regulated. Overexpression and activation of MMPs are correlated with a number of pathologies.

The composition of collagen in the aneurysm wall of men and women

Background: Loss of vessel wall integrity by degradation is essential for the development of abdominal aortic aneurysm (AAA) and ultimately its rupture. The observed greater rupture rate in women with AAA might be related to gender differences in the biomechanical properties of the aneurysm wall. The aim of the study was to compare the biomechanically important structure of collagen between men and women with AAA. Methods: Biopsies of the aneurysm walls were obtained during elective open repair of men (n = 14) and women (n = 14) treated for AAA. High‐performance liquid chromatography (HPLC), Western blot, messenger RNA expression, and histochemical analyses were performed to assess the cross‐linking and the amount and the composition of collagen. Results: There was neither a difference in the thickness of the aneurysm wall, nor in the histological evaluation of the collagen composition between the sexes. Relative collagen content in the aneurysm wall was similar in men and women, as assessed by messenger RNA expression and HPLC. Collagen cross‐linking differed between the sexes; women had more lysyl pyridinoline (LP) than men (0.140 vs 0.07; P = .005), resulting in a lower hydroxyl pyridinoline (HP):LP ratio (3.28 vs 8.41; P = .003). There was no difference in messenger RNA and protein expressions of lysyl hydroxylase and lysyl oxidase to associate with the lower HP:LP ratio in women. Conclusions: The composition of collagen in the aneurysm wall of men and women are in several aspects similar, with the exception of collagen cross‐linking, suggesting that the difference in rupture rate between the sexes rather depend on the composition of other vessel wall structures. Clinical Relevance: The marked differences in prevalence and rupture risk of abdominal aortic aneurysm between men and women suggest gender to be of importance for both aneurysm development and progression. To study the amount and composition of collagen in men and women is of great importance for the understanding of the degenerative process occurring in aneurysms in both sexes and how it potentially differs, in regards to the increased rupture rate observed in women.

Role of Endothelial Plasminogen Activators in Fibrinolysis and Repair-Associated Angiogenesis

Fibrin is a temporary matrix, which is formed after wounding a blood vessel and when plasma leaks from blood vessels forming a fibrous exudate, often seen in areas of inflammation and in tumors (Dvorak et al., 1992). The fibrin matrix not only acts as a barrier preventing further blood loss, but also provides a structure in which new microvessels can infiltrate during wound healing. Proper timing of the outgrowth of microvessels as well as the subsequent (partial) disappearance of these vessels is essential to ensure adequate wound healing and to prevent the formation of scar tissue. It is generally believed that plasminogen activators play an important role in the migration and invasion of leukocytes and endothelial cells, and in the dissolution of the fibrin matrix (Kwaan, 1966; Pepper et al., 1990; Koolwijk et al., 1996). Plasminogen activators are serine proteases, which enzymatically convert the zymogen plasminogen into the active protease plasmin, the prime protease that degrades fibrin. The production of plasminogen activators by endothelial cells not only contributes to the proteolytic events related to the formation of microvessels in a wound, but also plays a crucial role in the prevention of thrombosis. If fibrin becomes deposited within the lumen of a blood vessel, cessation of the blood flow may occur accompanied by ischemia and eventually death of the distal tissues. The endothelium contributes considerably to the maintenance of blood fluidity by exposing anticoagulant molecules, by providing factors that interfere with platelet aggregation, and by its ability to stimulate fibrinolysis. Lysis of intravascularly generated fibrin must occur rapidly. However, it should be limited to a local area, because a general elevation of fibrinolysis upon wounding would result in recurrent bleeding.

Proteases and Angiogenesis. Regulation of Plasminogen Activators and Matrix Metalloproteases by Endothelial Cells

Angiogenesis, the outgrowth of new blood vessels from existing ones, is an essential process during development, but this normally stops when the body becomes adult. In the absence of injury, overt angiogenesis in adults is limited to the reproductive system of females (formation of corpus luteum and placenta). However, the formation of new blood vessels is an essential factor in tissue repair (formation and regression of granulation tissue), which is necessary to restore healthy tissue after wounding and/or inflammation, and is associated with many pathological conditions, such as tumor development and rheumatoid arthritis (Folkman and Klagsbrun, 1987; Liotta et al., 1991; Folkman and Shing, 1992; Montesano, 1992; Colville-Nash and Scott 1992). Fibrin (Dvorak et al., 1992), inflammatory cells (Polverini, 1989) and angiogenic factors (Broadley et al 1989; Klagsburn and D’Amore, 1991; Shweiki et al., 1992; Plate et al., 1992; Sengeret al., 1993; Koch et al., 1994) are commonly observed in angiogenesis associated with disease in man. A series of sequential events can be distinguished during the formation of new microvessels: (i) degradation of the vascular basement membrane and the fibrin or interstitial matrix by endothelial cells; (ii) endothelial cell migration; (iii) endothelial proliferation; and (iv) the formation of new capillary tubes and a new basement membrane (Folkman, 1986).