A novel primate model of delayed wound healing in diabetes: dysregulation of connective tissue growth factor.

AIMS/HYPOTHESIS: Chronic non-healing wounds are a common complication of diabetes. Prolonged inflammation and decreased matrix accumulation may contribute. Connective tissue growth factor (CTGF) is induced during normal wound healing, but its regulation in diabetic wounds is unknown. We developed a primate model for the study of in vivo wound healing in baboons with long diabetes duration. METHODS: Drum implants were placed subcutaneously into thighs of diabetic and non-diabetic control baboons. After 2 and 4 weeks the skin incision sites were removed for measurement of breaking strength and epithelial thickness. Drum implants were removed for analysis of granulation tissue and inflammatory cells, CTGF and tissue inhibitor of matrix metalloproteinase (TIMP-1). Degradation of added CTGF by wound fluid was also examined. RESULTS: Healed incision site skin was stiffer (less elastic) in diabetic baboons and epithelial remodelling was slower compared with controls. Granulation tissue from diabetic baboons was reduced at 2 and 4 weeks, with increased vessel lumen areas at 4 weeks. Macrophages were reduced while neutrophils persisted in diabetic tissue. In diabetic wound tissue at 4 weeks there was less CTGF induced, as shown by immunohistochemistry, compared with controls. In contrast, immunoreactive fragments of CTGF were significantly increased in whole tissue lysate in diabetic baboons, suggesting that CTGF is redistributed in diabetes from granulation tissue into wound fluid. When recombinant human CTGF was co-incubated with wound fluid, increased CTGF degradation products were observed in both control and diabetic samples. CONCLUSIONS/INTERPRETATION: This baboon model of wound healing reflects the abnormal microenvironment seen in human diabetic wounds and provides insights into the dysregulation of CTGF in diabetic wounds.

Effects of pulsed electromagnetic field vibration on tooth movement induced by magnetic and mechanical forces: a preliminary study.

BACKGROUND: This study was designed to determine whether or not high-frequency and low-magnitude vibration affects orthodontic tooth movement caused by magnetic or/and mechanical forces. METHODS: Forty-four 7-week-old Wistar rats were randomly divided into four groups, with each group further divided into experimental and control subgroups. Neodymium-Iron-Boron (Nd-Fe-B) magnets and Sentalloy closed coil springs were placed between maxillary or mandibular first molars and incisors to activate tooth movement. The animals of experimental subgroups were exposed to the vibration induced by pulsed electromagnetic fields (PEMF) whilst the control subgroups were under normal atmosphere. The experiment lasted for 14 days and all of the animals were sacrificed for examination. The changes in the space between the molar and incisor were measured to indicate the amount of tooth movement. RESULTS: The coil springs, either with sham or active magnets, move molar much more than magnets alone, regardless of absence or presence of PEMF (p < 0.001). Under PEMF, the coil spring moved significantly more amount of tooth movement than that of coil-magnet combination (p < 0.01), as did the magnets compared to sham magnets (p < 0.019). Under a non-PEMF scenario, there was no significant difference in tooth movement between coil spring and coil-magnets combination, nor was there difference between magnets and sham magnets. CONCLUSIONS: It is suggested that the PEMF-induced vibration may enhance the effect of mechanical and magnetic forces on tooth movement.

Cytokine regulation of granulocyte-macrophage colony stimulating factor and macrophage colony-stimulating factor production in human arterial smooth muscle cells.

Smooth muscle cells (SMC) are the major cell type found in the walls of large blood vessels and appear to participate in local immune and inflammatory reactions, as well as in certain vascular diseases. We tested whether human arterial SMC can produce in vitro the colony stimulating factors (CSFs), granulocyte macrophage-CSF (GM-CSF) and macrophage CSF (M-CSF). Untreated internal mammary artery and aortic SMC produced no detectable GM-CSF but constitutively made M-CSF, measured by ELISA and radioimmunoassay, respectively. Interleukin-1 (IL-1) and, to a lesser extent, tumor necrosis factor alpha (TNF alpha) stimulated GM-CSF formation within 3 h; mRNA levels also increased particularly in the presence of the protein synthesis inhibitor, cycloheximide. IL-1, TNF alpha and, in addition, interferon-gamma (IFN-gamma) raised the M-CSF levels within 6 h; cycloheximide potentiated the effects of IL-1 and TNF alpha on mRNA levels. These results suggest that cytokine-stimulated human arterial SMC may be a source of the M-CSF found in atherosclerotic lesions. Since monocytes/macrophages can be activated by GM-CSF and M-CSF, while GM-CSF can also affect granulocyte function, SMC may participate in inflammatory reactions and vascular diseases by releasing these cytokines.

Histochemical identification of the vascular endothelial isoenzyme of alkaline phosphatase.

Alkaline phosphatase (AP) is a widely studied membrane bound ecto-enzyme with an extensive distribution in nature. Three major human isoenzymes have been defined and can be distinguished on the basis of their differential sensitivity to specific inhibitors. Despite the voluminous literature describing AP, the physiological role of this enzyme is unclear. Microvascular endothelium is strongly AP positive and may provide a convenient model for study of the role of AP in vitro. This report describes the use of freeze-substitution and high-resolution plastic embedding techniques to identify the isoenzyme of endothelial AP by quantitative analysis of the relative inhibition by specific inhibitors of AP, using human gingival tissues and a number of rat tissues. Endothelial AP is found to be the liver/bone/kidney isoenzyme, indicating kidney as a credible source of enzyme for further experimental work investigating the role of AP.

Cytokine regulation of the synthesis of plasminogen activator inhibitor-2 by human vascular endothelial cells. Comparison with plasminogen activator inhibitor-1 synthesis.

Plasminogen activators are inhibited by plasminogen activator inhibitors-1 (PAI-1) and -2 (PAI-2). We describe the synthesis of PAI-2 by human vascular endothelial cells (EC) cultured from umbilical vein, saphenous vein and foreskin microvasculature in response to interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor alpha (TNF alpha) and compare it with that of PAI-1. Both PAI-2 and PAI-1 were quantitated by ELISAs. PAI-2 was cell-associated while PAI-1 was secreted by EC. IL-1 alpha and TNF alpha increased the synthesis of PAI-2 and PAI-1 by EC in a dose-dependent manner. IL-1 alpha was a stronger stimulus for PAI-2 synthesis than TNF alpha, while both cytokines were equally effective for PAI-1. Northern blot analysis revealed similar changes in mRNA levels to those in antigen levels. PAI-2 synthesis by cytokine-stimulated EC may be important in thrombus formation and inflammation.

Human endothelial cells maintain anti-aggregatory activity for platelets during apoptosis.

Despite evidence of elevated levels of tissue factor and platelet binding by apoptotic endothelial cells, microthrombi do not appear to be associated with apoptotic endothelium and this suggests maintained anti-aggregatory activity for platelets. We report that anti-aggregatory activity is maintained by apoptotic endothelium obtained by serum and or matrix deprivation, which we propose as models for apoptotic endothelial cells released during microvascular remodelling and traumatic detachment respectively. Both apoptotic and non-apoptotic endothelium had strong anti-aggregatory activity for platelets stimulated with either ADP or thrombin. Inhibition experiments using L-NAME and indomethacin indicated a role for nitric oxide and prostacyclin in this activity. Experiments with latex beads further confirmed that inhibited platelet aggregation by endothelium was not merely a non-specific phenomenon. These data support the idea that EC maintain active antithrombotic activity during apoptosis, consistent with maintained urokinase levels and canalicular fragmentation reported elsewhere.

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells.

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.

Fibrinolytic proteins in apoptotic human umbilical vein endothelial cells.

Endothelial cells express fibrinolytic proteins including: urokinase (u-PA) and tissue type (t-PA) plasminogen activators, type-1 (PAI-1) and 2 (PAI-2) plasminogen activator inhibitors, and u-PA receptor (u-PAR). Apoptotic endothelial cells detach, potentially forming both local and circulating microthrombi in vivo. In this article, apoptotic human umbilical vein endothelium was obtained by serum starvation and compared with nonapoptotic cells rescued from death with fresh medium containing serum. Antigen levels for t-PA, PAI-1, PAI-2, and u-PAR were reduced greatly in apoptosis (p< 0.05). In contrast, u-PA levels were similar in apoptotic as compared with rescued cells (p<0.05). Radioactive amino acids were used to determine absolute levels of protein synthesis and degradation in these cells. Reduced antigen levels likely were due to proteolysis as there was 98% total protein degradation and very little protein synthesis in apoptotic endothelial cells. Also, u-PA levels in apoptotic endothelial cells were not affected by the protein synthesis inhibitor cycloheximide. Endothelial cells in inflammatory sites are exposed to cytokines, which increase both apoptosis and u-PA levels. Data from this article support the idea that maintained u-PA levels in apoptotic endothelium may protect from micro-thrombosis in inflammatory sites as well as in the circulation.

Chronic adult periodontitis and burst progression may reflect local neutrophil defects due to perivascular hyaline deposits.

Chronic adult periodontitis (CAP) is a common disease of the supporting tissues of teeth, and is a major cause of tooth loss. This disease is distinguished from more rare rapidly progressing forms of periodontitis, in which a variety of neutrophilic polymorphonuclear leukocyte (PMN) defects have been identified. PMN dysfunctions have, however, not been observed in CAP. In CAP, destructive episodes of the disease occur sporadically and independently in different parts of the mouth. In this paper, it is proposed that CAP is due to highly localized defects in PMN function. Impaired PMN function is suggested as resulting in the formation of a virulent bacterial plaque, which is capable of initiating periodontal pocket formation. A previously reported perivascular hyaline material may account for localized PMN defects, by reducing the number of PMNs entering affected sites. The proposed model may explain both the presence of CAP in otherwise normal patients, and the sporadic pattern of tissue destruction seen in this disease.

The vascular response in chronic periodontitis.

The role of vascular endothelial cells (EC) in periodontitis was investigated in a series of histological studies. Expansion of the vasculature was found to occur with development of gingivitis and periodontitis. This was thought to contribute to the characteristic tissue degradation in the developing disease. Vascular expansion could also play a role in the formation of a previously unreported perivascular hyaline material (PHyM). Polymorphonuclear leukocytes (PMN) are known to be protective in periodontitis, and the location, incidence and extent of PHyM suggested a role for PHyM in periodontitis by inhibiting PMN emigration. PMN emigration was found to occur from specialized high EC (HEC) lined post capillary venules. This was unexpected, as such vessels have previously been found to exchange lymphocytes almost exclusively. Detailed histochemical, ultrastructural and biosynthetic studies of these specialized blood vessels led to the suggestion that HEC may be specially adapted for the synthesis of cytokines in periodontitis. A negative association between expression of the membrane bound ectoenzyme, alkaline phosphatase, and HEC suggested a role for this enzyme in leukocyte emigration. These observations compel re-evaluation of the role of EC in chronic inflammation, and in periodontitis in particular. The direction of current and future work is discussed.

Vascular expansion in chronic periodontitis.

Animal studies have demonstrated expansion and remodelling of gingival blood vessels in inflamed gingival tissues. However, there is a paucity of information, regarding the vascular response in human gingivitis and periodontitis. In this study, gingival biopsies were obtained from 51 separate patients. Fifteen minimally inflamed, 16 gingivitis and 20 periodontitis specimens were studied. Gingival biopsies were divided into five fields, and a quantitative survey of vascular changes was performed. In fields adjacent to the bacterial plaque irritant, vessel profiles were increased in number with the development of the advanced periodontal lesion. The diameter of blood vessels throughout the entire thickness of gingival biopsies was found to increase with advancing periodontal disease. It is concluded that considerable remodelling of the gingival vasculature occurs in chronic periodontitis, and that this may contribute to the tissue destruction seen in this disease.

Perivascular hyaline deposits in inflamed gingival tissues.

A perivascular hyaline material (PHyM) was found in gingival biopsies from patients with periodontitis, gingivitis and minimally inflamed gingiva. PHyM was found only in association with the sulcular or pocket epithelium. The extent and frequency of the deposits was quantitatively associated with inflammation of the gingival tissues, as well as with the apical region of periodontal pockets. Evidence for angiogenesis was found in association with the deposition of PHyM. The ultrastructure of the PHyM indicated that the material, which was of an amorphous hyaline appearance at the light microscope level, was composed of multiple basal lamina impregnated with irregular collagen fibrils, fine fibrils and cellular debris. The basal lamina material was degraded at many sites. Immunohistochemistry confirmed the abundance of type IV collagen, supporting the basal lamina origin for PHyM. It is proposed that the deposition of the hyaline matrix is related to the effect of angiogenic and injurious agents on the vascular endothelium. PHyM could contribute to the development of periodontitis by impairing the emigration of polymorphonuclear leukocytes into the gingival sulcus.

High endothelial-like venules in chronically inflamed periodontal tissues exchange polymorphs.

A survey of 58 gingival biopsies revealed the presence of periodontal high endothelial-like venules (PHELVs) in chronically inflamed gingival tissues. PHELVs were found to exchange polymorphonuclear cells (PMNs) almost exclusively in advanced periodontitis, with PMNs greatly exceeding the number of mononuclear cells found in PHELVs (P less than 0.001). Electron microscopy confirmed the emigration of PMNs from these vessels. The enzyme histochemical and ultrastructural features as well as the 35SO4 uptake properties of PHELVs were similar to those of the well-characterized high endothelial venules (HEVs) of rat lymph nodes. It is generally accepted that HEVs in lymphoid tissues and inflammatory sites are specially adapted to assist in the emigration of lymphocytes. However, the observation of preferential PMN emigration in the apparent absence of lymphocyte exchange from PHELVs compels further investigation of other possible functions for HEVs. In relation to this, endothelial cells are capable of producing potent cytokines and inflammatory mediators which may contribute to the development of lesions, and the possibility is discussed that high endothelial cells are functionally adapted to enhance the production of such factors.

Association of amyloid P protein with pathology in periodontal tissues.

The lesion of chronic periodontitis is characterized by the persistence of perivascular collections of degenerate plasma cells. In this study, immunohistochemical demonstration of amyloid P (AP) component was used to define the distribution of this protein in established periodontitis lesions and in biopsies of non-destructive marginal gingivitis. Quantitative assessment of AP indicated significantly higher levels in periodontitis than in gingivitis for all regions of the tissue. This was associated with pathology as determined by the intensity of plasma cell accumulation and the extent of connective tissue matrix degradation. AP was concentrated in the deep connective tissue areas but perivascular accumulation was also noted, as was deposition associated with nerve bundles and, occasionally, in the extracellular matrix of the lining epithelium. These findings have potential significance in relation to the pathology of chronic periodontitis as AP has been shown to interact in a calcium-dependent manner with a number of ligands including fibronectin, elastic fibres, C-4 binding protein and amyloid fibrils.

Extraction of amyloid-like fibrils from chronically inflamed periodontal tissues.

Immunohistological studies have established an association between the deposition of the amyloid P protein and disease status in chronically inflamed periodontal tissues. The aim of this study was to determine if amyloid-like fibrils could be extracted from these tissues. Biopsies were homogenised and extracted exhaustively in saline before serial extraction in distilled water. Electrophoretic analysis revealed the presence of previously undetected protein bands in the fifth water extraction. These were probed and were found to react with antisera to kappa and lambda immunoglobulin light chains but not with antisera to mu, gamma or alpha heavy chains. Electron microscopic study indicated fibrils of 9.7 nm diameter. These bound Congo Red and exhibited green birefringence under polarised light. The results supported the presence of an amyloid-like matrix composed of immunoglobulin light chains in the lesions of chronic periodontitis. This could explain the persistence of foci of degenerate plasma cells and the paucity of granulation tissue formation in the disease process.

Contrasting effects of interleukin-4 on colony-stimulating factor and interleukin-6 synthesis by vascular endothelial cells.

Endothelial cells (EC) may regulate both local and systemic aspects of inflammation through the synthesis of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-6 (IL-6). EC are known to synthesize these cytokines in response to interleukin-1 (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS). In this paper, we illustrate the effect of interleukin-4 (IL-4) in reducing the synthesis of GM-CSF by EC stimulated with IL-1 alpha, TNF-alpha, or LPS. This is compared with the previously reported strong synergy between IL-4 and IL-1 alpha, TNF-alpha, or LPS in the synthesis of IL-6 by EC. No clear effect of IL-4 was seen in the synthesis of G-CSF or M-CSF. The range of concentrations of IL-4 at which these effects were seen was identical for both reduced GM-CSF synthesis and increased IL-6 synthesis. The effect of IL-4 on IL-6 synthesis was seen by 4 h of treatment, while that on GM-CSF was apparent between 4 and 8 h. It is suggested that these contrasting effects of IL-4 may reflect a biological role for this cytokine in the regulation of leukocytosis and the acute phase response.

Induction of contact-dependent endothelial apoptosis by osteosarcoma cells suggests a role for endothelial cell apoptosis in blood-borne metastasis.

Although tumour cells are believed to migrate between endothelial cells early in metastasis, the possibility remains that endothelial apoptosis may also contribute to the tumour's breach of the vascular barrier. Although seemingly inconsistent with tumour angiogenesis, one publication describes the induction of contact-dependent apoptosis in cultured endothelium by tumour cells. The cell culture data are, however, open to challenge on technical grounds while there are no confirmatory reports. The present paper describes experiments overcoming these limitations. SAOS-2 human osteosarcoma cells and two rat carcinoma cell lines were co-cultured with human umbilical vein endothelial cells (HUVECs) and cultures labelled by surface lectin histochemistry for endothelium. The HUVEC culture density was determined and SAOS-2 cells, but not rat carcinoma cells, were found significantly to reduce HUVEC survival despite the release of potent growth factors as determined in separate experiments with tumour cell conditioned medium. Lectin labelling combined with light microscopy, transmission electron microscopy, flow cytometry for both lectin binding and DNA content, and DNA gel electrophoresis of SAOS-2/HUVEC co-cultures revealed extensive HUVEC apoptosis. These findings indicate contact-dependent endothelial apoptosis by SAOS-2, while this activity appeared weaker and overwhelmed by HUVEC proliferation with rat carcinoma cells. Importantly, this study supports the suggestion that endothelial apoptosis may be important for metastasis and suggests a complex interplay between endothelial proliferation and apoptosis in tumours.

Interferon-alpha 2 counteracts interleukin-1 alpha-stimulated expression of urokinase-type plasminogen activator in human foreskin microvascular endothelial cells in vitro.

We investigated the effect of interferon-alpha 2 (IFN-alpha 2) on interleukin-1 alpha (IL-1 alpha)-induced up-regulation of urokinase type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and human umbilical vein endothelial cells (HUVEC) in vitro. When IFN-alpha 2 and IL-1 alpha were added to the cells simultaneously, IFN-alpha 2 inhibited IL-1 alpha-induced up-regulation of u-PA antigen in a dose- and time-dependent fashion in HFMEC, whereas in HUVEC no effect of IFN-alpha 2 on IL-1 alpha-induced u-PA was seen. IL-1 alpha-induced up-regulation of PAI-1 antigen in HFMEC was not counteracted by IFN-alpha 2. When IFN-alpha 2 was added to HFMEC 1 or 2 h after IL-1 alpha a significant inhibition in u-PA synthesis was seen, whereas when IFN-alpha 2 was added to the cells 8 h after IL-1 alpha no effect on the induction of u-PA synthesis by IL-1 alpha was seen. IFN-alpha 2 also inhibited significantly the IL-1 alpha stimulated up-regulation of specific u-PA mRNA expression. In conclusion, our data show that IFN-alpha 2 can counteract the IL-1 alpha-induced up-regulation of u-PA in a similar way as IFN-gamma. This effect, which seems to be specific for microvascular endothelial cells, could contribute to the modulation of endothelial cell-mediated extravascular proteolysis in processes such as wound healing, neovascularisation, and endothelial cell migration.

Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells.

The effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase-type plasminogen activator (u-PA) (control: 3.0 +/- 0.8 ng/10(5) cells/24 h; 200 U/mL IL-4: 6.7 +/- 0.8 ng/10(5) cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 +/- 0.003 IU/10(5) cells/20 h; 200 U/mL IL-4: 0.09 +/- 0.007 IU/10(5) cells/20 h). These values increased to 0.18 +/- 0.02 IU/10(5) cells/20 h and 0.53 +/- 0.04 IU/10(5) cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting. u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.

Human arterial smooth muscle cells synthesize granulocyte colony-stimulating factor in response to interleukin-1 alpha and tumor necrosis factor-alpha.

Vascular smooth muscle cells (SMC) are a major cell type comprising the walls of blood vessels. We report the synthesis of granulocyte colony-stimulating factor (G-CSF) by cultured human SMC obtained from the internal mammary artery and thoracic aorta. Interleukin-1 alpha (IL-1 alpha) greatly increased in a dose-dependent manner the amount of this cytokine produced by the SMC, with tumor necrosis factor-alpha (TNF-alpha) being less effective. Newly formed G-CSF could be detected in culture supernatants within 6 hours after IL-1 alpha or TNF-alpha treatment. Northern blot analysis of SMC stimulated with IL-1 alpha and TNF-alpha showed an increase in the amount of mRNA for G-CSF as compared with control cells. Enhanced G-CSF mRNA levels were observed when SMC were treated with cycloheximide in the absence or presence of added cytokine. In vasculitis, the walls of blood vessels become inflamed as evidenced by a leucocytic infiltrate usually dominated by polymorphonuclear neutrophil leukocytes (PMNs). G-CSF is known to stimulate PMNs, and our findings raise the possibility that G-CSF made by SMC contributes to the development of vasculitis lesions.