Matrix metalloproteinases and failed fracture healing

During fracture healing and the resulting formation of new bone, an extensive amount of extracellular matrix is synthesized which subsequently undergoes enzymatic remodeling and then mineralization. The remodeling process of mostly collagenous molecules is largely attributable to matrix metalloproteinases (MMPs). A variety of members of this protease family and its respective inhibitors – termed tissue inhibitors of matrix metalloproteinases (TIMP) – have been found to be closely related to the fracture healing process. Delays in bone healing or even nonunion could be related to the concentrations of these enzymes or their behavior over time. In this study, serum samples were prospectively collected from patients who had undergone surgical treatment for limb fracture. Serum probes from 15 patients with nonunion of fractures 4 months after surgery have been compared to 15 matched patients with normal bone healing. Postoperative time courses of serum concentrations of MMP-1/-2/-3/-8/-9/-13 as well as TIMP-1/-2 were analyzed using commercially available enzyme immunoassays.Comparison between both collectives revealed significantly elevated serum concentrations of proMMP-1 in the nonunion group at 2 and 24 weeks after surgery. Similar findings were found for MMP-8 at 2, 4 and 8 weeks. At 1 week after surgery, TIMP-1 serum concentrations were significantly lower in nonunion patients when compared to patients with normal bone repair.We have been able to show for the first time the course of serum concentrations of MMPs and TIMPs during normal and delayed fracture healing. Characteristic time courses of systemic MMP- and TIMP-levels could be a reflection of local enzyme regulatory mechanisms during fracture healing. An altered balance of the MMP/TIMP system in favor of proteolytic activity as shown in our investigation may be involved in the pathophysiological processes leading to fracture nonunion.     

Matrix metalloproteinases and failed fracture healing

During fracture healing and the resulting formation of new bone, an extensive amount of extracellular matrix is synthesized which subsequently undergoes enzymatic remodeling and then mineralization. The remodeling process of mostly collagenous molecules is largely attributable to matrix metalloproteinases (MMPs). A variety of members of this protease family and its respective inhibitors – termed tissue inhibitors of matrix metalloproteinases (TIMP) – have been found to be closely related to the fracture healing process. Delays in bone healing or even nonunion could be related to the concentrations of these enzymes or their behavior over time. In this study, serum samples were prospectively collected from patients who had undergone surgical treatment for limb fracture. Serum probes from 15 patients with nonunion of fractures 4 months after surgery have been compared to 15 matched patients with normal bone healing. Postoperative time courses of serum concentrations of MMP-1/-2/-3/-8/-9/-13 as well as TIMP-1/-2 were analyzed using commercially available enzyme immunoassays.

Comparison between both collectives revealed significantly elevated serum concentrations of proMMP-1 in the nonunion group at 2 and 24 weeks after surgery. Similar findings were found for MMP-8 at 2, 4 and 8 weeks. At 1 week after surgery, TIMP-1 serum concentrations were significantly lower in nonunion patients when compared to patients with normal bone repair.

We have been able to show for the first time the course of serum concentrations of MMPs and TIMPs during normal and delayed fracture healing. Characteristic time courses of systemic MMP- and TIMP-levels could be a reflection of local enzyme regulatory mechanisms during fracture healing. An altered balance of the MMP/TIMP system in favor of proteolytic activity as shown in our investigation may be involved in the pathophysiological processes leading to fracture nonunion.     

Increased serum OPG in atrophic nonunion shaft fractures

Background  Bone repair alteration is hypothesized for nonunion fracture pathogenesis. Since it is involved in osteoclast regulation, the RANK/RANKL/OPG system (receptor activator of nuclear factor kB/its ligand/osteoprotegerin) may play a role. Materials and methods  Serum OPG, free RANKL, bone alkaline phosphatase (BAP), osteocalcin (OC), and urinary deoxypyridinoline (DPD) were determined in 16 male patients (20–39 years) with long bone atrophic nonunion fractures. Serum markers were also measured in 18 age-matched male controls who healed from the same type of fractures within six months, and in 14 age-matched male controls who were healing from the same fractures one month after injury. One-way ANOVA and Bonferroni’s test were used for statistical analysis. Results  Only OPG was significantly higher (0.56 sd 0.11 ng/ml) in the patients compared to healed (0.26 sd 0.04 ng/ml; P < 0.001) and healing (0.29 sd 0.09 ng/ml; P < 0.001) controls. The patients’ DPD levels were normal. No correlations were found between bone markers and the characteristics of the subjects in all groups. Conclusions  A normal steady state of bone metabolism seems to be present in patients with atrophic nonunion fractures, despite the high serum OPG. The reason for the inability of the patients’ OPG to inhibit osteoclastic activity is unknown. Osteoblast activity also appears normal, so another cellular source of OPG can be hypothesized.     

Quantification of TGF-ß1, PDGF and IGF-1 cytokine expression after fracture treatment vs. non-union therapy via masquelet

IntroductionBiochemical processes during bone regeneration can be analysed via quantification of peripheral serum cytokine levels. To date, serum levels of cytokines in patients treated with masquelet technique and patients with normal bone healing have not been compared. This comparison is supposed to deliver novel insights into the process of bone regeneration. Our aim was to validate this established method in the monitoring of bone regeneration after non-union treatment in masquelet technique.Materials and methodsBetween 04/2008 and 01/2014 three groups were recruited: G1 (10 patients) with long bone non-unions, treated successfully with masquelet therapy, G2 (6 patients) with unsuccessful masquelet therapy and G3 (10 patients) with long bone fractures and normal bone healing. Peripheral blood samples were collected over a period of six months following a standardised time pattern in combination with clinical and radiologic follow up. TGF-ß1, PDGF-AB and IGF-1 were measured using commercially available immunoassays.ResultsTGF-ß1 levels in G1 and G2 demonstrated a parallel and lower overall concentration over time compared to G3. G3 showed a significant TGF-ß1 peak 2 weeks after surgery compared to G1 (p = 0.0054). PDGF-AB concentrations were always lower in G2 than in G1 and G3. G3 peaked at week 2 with a significant higher value than in G2 (p = 0.0177). IGF-1 showed lower overall serum concentrations in G2 than in G1 and G3. G1 had a peak level during the fourth week of follow-up. Compared to G2 this peak was significant (p = 0.0015).ConclusionsThis study shows that successful bone regeneration via masquelet technique only partially imitates cytokine expression of physiological bone healing. High expressions of IGF-1 correspond to a successful masquelet therapy while TGF-ß seems to play a minor role. These results assume that objective analysis of an effective non-union therapy with cytokine expression analysis is possible even with a small number of patients.     

Prognostic Significance of Vascular Endothelial Growth Factor,Basic Fibroblast Growth Factor,and Angiogenin in Patients With Resectable Hepatocellular Carcinoma After Surgery

Background: Hepatocellular carcinoma (HCC) is a hypervascular malignancy. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin (ANG) are important angiogenic factors of neoangiogenesis. This study investigated the predictive value of serum VEGF, bFGF, and ANG in tumor recurrence, disease-free survival (DFS), and overall survival (OS) in HCC patients.Methods: Preoperative serum VEGF, bFGF, and ANG were measured in 98 patients with resectable HCC and in 15 healthy controls. The median follow-up time was 43 months.Results: Preoperative serum VEGF was increased in patients with resectable HCC compared with healthy controls (P < .05). Increased serum VEGF was correlated with tumor recurrence (P = .001). Univariate analysis showed that serum VEGF, tumor-node-metastasis stage, tumor size and number, macroscopic portal vein invasion, and microscopic vascular invasion were correlated with OS and DFS. Serum bFGF and ANG were not associated with survival. Multivariate analysis showed that serum VEGF was the most significant predictor of DFS (relative risk, 2.35; 95% confidence interval, 1.26–4.39; P = .007) and OS (relative risk, 3.44; 95% confidence interval, 1.81–6.57; P < .001) in HCC patients after surgical resection.Conclusions:Preoperative serum VEGF is a significant independent predictor of tumor recurrence, DFS, and OS in patients with resectable HCC.     

Base Deficit ≥ 6 within 24 h of Injury is a risk factor for fracture nonunion in the polytraumatized patient

BackgroundPolytrauma patients are at risk for fracture nonunion, but the reasons are poorly understood. Increased base deficit (BD) is associated with hypovolemic shock. Although shock delays bone healing in animal models, there have been no clinical studies evaluating the impact of BD on nonunion risk.Materials and MethodsPatients age ≥ 16 with injury severity score > 16 that presented to an academic Level One trauma center with an operative femur or tibia fracture were reviewed. Clinical notes and radiographs were assessed to determine fracture healing status. Patient demographics, injury characteristics, BD, and number of packed red blood cell transfusions were recorded. Bivariate and multivariate analyses of multiple risk factors associated with nonunion were conducted to investigate the association of BD with nonunion.ResultsThe union group was comprised of 243 fractures; there were 36 fractures in the nonunion group. The following predictors were associated with nonunion: smoking (p = 0.009), alcohol use (p < 0.001), open fracture (p < 0.001), and treatment for deep infection at fracture site (p = 0.016). Additionally, worst BD over 24 h ≥ 6 (p = 0.031) was significant for nonunion development. A multivariate logistic regression analysis revealed worst BD ≥6 over 24 h remained significantly associated with the development of nonunion (odds ratio 3.02, p = 0.011) when adjusting for other risk factors.ConclusionsA BD ≥ 6 within 24 h of admission was associated with a significantly increased risk of developing lower extremity fracture nonunion in polytrauma patients, even after adjusting for multiple other risk factors. Acute post-traumatic acidosis may have effects on long-term fracture healing.     

TRACP 5b and CTX as osteological markers of delayed fracture healing

Radiological studies are the standard method to monitor fracture healing but they do not allow a timely assessment of bone healing. Biochemical markers react rapidly to changes in bone metabolism during fracture healing and could be an additional tool to monitor this process.The goal of this study was to observe changes in serum biomarkers and evaluate the possible differences in the serum levels of tartrate-resistant acid phosphatase 5b (TRACP 5b), total N-terminal propeptide of type I collagen (PINP), bone-specific alkaline phosphatase (BAP), and C-terminal cross-linking telopeptide of type I collagen (CTX) in patients with normal and delayed fracture healing.Several serum samples were collected for one year after the surgical treatment of long bone fractures in 248 patients. From this large pool, 15 patients with atrophic nonunion were matched to 15 patients with normal bone healing. Post-operative changes in osteological markers were monitored during the 1st, 2nd, 4th, 8th, 12th and 52nd weeks. The patients were followed both clinically and radiologically for the entire one-year duration of the study.In the first week, the absolute values of CTX decreased significantly (p = 0.0164) in cases of delayed fracture healing. The relative values of TRACP 5b were significantly decreased at weeks 4 (p = 0.0066) and 8 (p = 0.0043). BAP and PINP levels decreased in the first week followed by an increase, but there were no significant differences in the absolute or relative values during the healing process in both patient groups.For the first time, we have demonstrated changes in serum concentrations of TRACP 5b, PINP, BAP, and CTX during normal and delayed fracture healing. Characteristic changes in systemic TRACP 5b and CTX levels could reflect the initial process of successful fracture healing and may be used in clinical practice to monitor the healing process. Furthermore, it could be very important for determining the beneficial effects of additional treatments such as ultrasound or BMPs in clinical trials.     

Elevated transforming growth factor-beta 1 (TGF-β1) levels in human fracture healing

Introduction

Transforming growth factor-beta 1(TGF-β1) is a regulatory protein, involved in bone fracture healing. Circulating TGF-β1 levels have been reported to be a predictor of delayed bone healing and non-union, suggesting active relationship between tissue and circulating TGF-β1 in fracture healing. The purpose of this study was to analyse TGF-β1 local and serum concentrations in fracture healing to further contribute to the understanding of molecular regulation of fracture healing.

Patients and methods

Serum samples of 113 patients with long bone fractures were collected over a period of 6 months following a standardised time schedule. TGF-β1 serum concentrations were measured using ELISA. Patients were assigned to 2 groups: Group 1 contained 103 patients with physiological healing. Group 2 contained 10 patients with impaired healing. Patients in both groups were matched. One patient of the group 2 had to be excluded because of missing match partner. In addition, fracture haematoma from 11 patients of group 1 was obtained to analyse local TGF-β1 concentrations. 33 volunteers donated serum which served as control.

Results

TGF-β1 serum concentrations increased during the early healing period and were significantly higher in patients with physiological healing compared to controls (P = 0.04). Thereafter, it decreased continuously between weeks 2 and 8 and fell again after week 8. TGF-β1 serum concentrations in patients with physiological healing were significantly higher at week 24 compared to controls (P = 0.05). In non-unions, serum concentrations differed significantly from those of controls at week 6 (P = 0.01). No significant difference in between patients with physiological and impaired fracture healing was observed. Fracture haematoma contained significantly higher TGF-β1 concentrations than peripheral serum of the patients (P = 0.017).

Conclusion

Elevated levels of TGF-β1 in haematoma and in serum after bone fracture especially during the entire healing process indicate its importance for fracture healing.     

Angiopoietin-1, but not platelet-derived growth factor-AB, is a cooperative stimulator of vascular endothelial growth factor A-accelerated endothelial cell scratch closure

Wound healing and the grow-in of free tissue grafts critically depend on blood vessel growth, i.e., on the angiogenic invasion of endothelial cells, which is critically reduced in smokers, in patients suffering from microangiopathies (e.g., in diabetes), or in those who are treated with immunosuppressives. Although several angiogenic factors have been tested to accelerate wound healing in such critically patients, their combinations have not yet been systematically investigated. This study was done to reveal which combination of proangiogenic with promaturating factors is the most effective in an endothelial wound closure assay. Human umbilical vein endothelial cells were isolated, cultured to confluence, and subjected to a scratch wound assay with the addition of vascular endothelial growth factor (VEGF)-A(165), platelet-derived growth factor (PDGF)-AB, angiopoietin-1 (ANG1), or ANG2 and all of their 16 possible combinations. VEGF-A(165) plus ANG1 was most effective at accelerating endothelial scratch closure. Moreover, VEGF-A(165) stimulated wound closure in all combinations tested, while it was attenuated by PDGF-AB. Thus, with respect to their effects on endothelial cells, a combination of VEGF-A with ANG1 is the most promising and is superior to combinations with PDGF-AB.     

Elevated levels of macrophage colony‐stimulating factor in human fracture healing

Macrophage colony‐stimulating factor (M‐CSF) plays a unique role in bone remodeling. However, to our knowledge, no data on the role of M‐CSF in fracture healing in humans have been published so far. This study addressed this issue. One hundred and thirteen patients with long‐bone fractures were included in the study and divided into two groups, according to their course of fracture healing. The first group contained 103 patients with normal fracture healing. Ten patients with impaired fracture healing formed the second group of the study. Volunteers donated blood samples as control. Serum samples were collected over a period of 6 months, following a standardized time schedule. In addition, M‐CSF levels were measured in fracture hematoma and serum of 11 patients with bone fractures. M‐CSF concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). Fracture hematoma contained significantly higher M‐CSF concentrations compared to M‐CSF concentrations in patient's serum. M‐CSF levels in fracture hematoma and in patient's serum were both significantly higher than M‐CSF concentrations measured in serum of healthy controls. Highly elevated M‐CSF serum concentrations were found in patients with physiological fracture healing over the entire observation period. Significant differences in the M‐CSF serum concentration between patients with normal fracture healing and patients with impaired fracture healing were not observed. This study indicates, for the first time, to our knowledge, a possible local and systemic involvement of M‐CSF in humans during fracture healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:671–676, 2010     

Strongly enhanced levels of sclerostin during human fracture healing

Sclerostin (SOST), an antagonist of Wnt signaling, is an important negative regulator of bone formation. However, no data on the role of SOST in the human fracture healing have been published so far. This study addressed this issue. Seventy‐five patients with long bone fractures were included into the study and divided in two groups. The first group contained 69 patients with normal fracture healing. Six patients with impaired fracture healing formed the second group. Thirty‐four volunteers donated blood samples as control. Serum samples were collected over a period of 1 year following a standardized time schedule. In addition, SOST levels were measured in fracture hematoma and serum of 16 patients with bone fractures. Fracture hematoma contained significantly higher SOST concentrations compared to patient's serum. SOST levels in fracture hematoma and in patient's serum were both significantly higher than in the serum of controls. Highly elevated SOST serum concentrations were found in patients with physiological fracture healing. SOST levels were decreased in patients with impaired fracture healing. However, this difference was not statistically significant. This is the first study to provide evidence of strongly enhanced SOST levels in patients with bone fracture. The results indicate local and systemic involvement of SOST in humans during fracture healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1549–1555, 2012     

Cigarette smoking decreases TGF-β1 serum concentrations after long bone fracture

TGF-β1 serum concentrations are considered to be one of the most promising markers of fracture healing. Previously, we demonstrated significant differences in the post-traumatic time courses of patients with timely and delayed fracture healing. The aim of this study was to evaluate possible differences in the serum concentrations of TGF-β1 in cigarette-smoking vs. non-smoking patients with timely and delayed fracture healing in order to understand pathophysiological pathways through which smoking impairs fracture healing.Serum samples were collected from 248 patients undergoing surgical treatment for long bone fractures within 1 year of surgery. Samples from 14 patients with atrophic-type delayed fracture healing were compared with 14 matched patients with normal bone healing. Each group included seven smokers and seven non-smokers. Post-operative serum concentrations were analysed at 1, 2, 4, 8, and 12 weeks as well as 1 year after surgery. The patients were monitored both clinically and radiologically for the entire duration of the study.All patients increased TGF-β1 serum concentrations after surgery. In patients with normal fracture healing, significantly higher TGF-β1 levels were observed in non-smokers (70 ng/ml) than in smokers (50 ng/ml) at the 4th week after surgery (p = 0.007). Also at the 4th week, in patients with delayed healing, significantly lower TGF-β1 levels were observed in smokers than in non-smokers (38 ng/ml vs. 47 ng/ml, p = 0.021). However, no significant differences between non-smokers with delayed healing and smokers with normal healing (p = 0.151) were observed at the 4th week after surgery. TGF-β1 serum concentrations reached a plateau in all groups from the 6th to the 12th week after surgery, with a slight decrease observed in the final measurement taken 1 year after surgery.This study demonstrates that, after fracture, TGF-β1 serum concentrations are reduced by smoking, and this reduction is statistically significant during the 4th week after surgery. Our findings may help reveal the mechanism by which smoking impairs fracture healing. Furthermore, these results may help to establish a serological marker that predicts impaired fracture healing soon after the injury. Surgeons will not only be able to monitor the bone healing, but they will also be able to monitor the success of additional treatments such as ultrasound and bone morphologic proteins (BMPs).     

Systemic regulation of angiogenesis and matrix degradation in bone regeneration—Distraction osteogenesis compared to rigid fracture healing

Aim of this study was the investigation of systemic biochemical regulation mechanisms of bone regeneration by angiogenic and matrix-degrading enzymes during distraction osteogenesis compared to rigid osteotomy bone healing.Serum samples of 10 otherwise healthy patients with callus distraction for lower limb-lengthening and 10 osteotomy patients undergoing elective axis correction have been collected prospectively in a standardized time schedule before and up to 6 months after the procedure. At the end of the individual investigation period, concentrations of metalloproteinases (MMP-9, -13), tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and the angiogenic factors angiogenin and VEGF have been detected by use of commercially available enzyme immunoassays. Results have been compared to our preliminary study on proMMP-1–3.In distraction osteogenesis, significantly elevated serum concentrations compared to baseline could be detected postoperatively for proMMP-1, MMP-9, TIMP-1, angiogenin and VEGF but not for proMMP-2, proMMP-3 or TIMP-2. In patients with rigid osteotomy healing, MMP-9, TIMP-1, TIMP-2, angiogenin and VEGF were significantly increased respectively. Comparison of both patient collectives revealed significantly higher increases of serum proMMP-1, VEGF and TIMP-1 in distraction patients during the lengthening period and significantly higher serum concentrations of TIMP-2 in late fracture healing period in osteotomy patients. Serum levels of MMP-13 were below the lowest standards, and therefore quantitative analysis was not possible. Bone regeneration in distraction osteogenesis and rigid osteotomy healing is accompanied by systemic increase of matrix-degrading and angiogenic factors in a certain time course and quantity. This might reflect biochemical regulation of local bone healing in the circulation. ProMMP-1, VEGF and TIMP-1 seem to be key regulatory factors during distraction osteogenesis.     

VEGF serum concentrations in patients with long bone fractures: A comparison between impaired and normal fracture healing

Vascular endothelial growth factor (VEGF) plays an important role in the bone repair process as a potent mediator of angiogenesis and it influences directly osteoblast differentiation. Inhibiting VEGF suppresses angiogenesis and callus mineralization in animals. However, no data exist so far on systemic expression of VEGF with regard to delayed or failed fracture healing in humans. One hundred fourteen patients with long bone fractures were included in the study. Serum samples were collected over a period of 6 months following a standardized time schedule. VEGF serum concentrations were measured. Patients were assigned to one of two groups according to their course of fracture healing. The first group contained 103 patients with physiological fracture healing. Eleven patients with delayed or nonunions formed the second group of the study. In addition, 33 healthy volunteers served as controls. An increase of VEGF serum concentration within the first 2 weeks after fracture in both groups with a following decrease within 6 months after trauma was observed. Serum VEGF concentrations in patients with impaired fracture healing were higher compared to the patients with physiological healing during the entire observation period. However, statistically significant differences were not observed at any time point between both groups. VEGF concentrations in both groups were significantly higher than those in controls. The present results show significantly elevated serum concentrations of VEGF in patients after fracture of long bones especially at the initial healing phase, indicating the importance of VEGF in the process of fracture healing in humans. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1293–1297, 2009     

Systemic regulation of angiogenesis and matrix degradation in bone regeneration--distraction osteogenesis compared to rigid fracture healing

Aim of this study was the investigation of systemic biochemical regulation mechanisms of bone regeneration by angiogenic and matrix-degrading enzymes during distraction osteogenesis compared to rigid osteotomy bone healing.

Serum samples of 10 otherwise healthy patients with callus distraction for lower limb-lengthening and 10 osteotomy patients undergoing elective axis correction have been collected prospectively in a standardized time schedule before and up to 6 months after the procedure. At the end of the individual investigation period, concentrations of metalloproteinases (MMP-9, -13), tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) and the angiogenic factors angiogenin and VEGF have been detected by use of commercially available enzyme immunoassays. Results have been compared to our preliminary study on proMMP-1–3.

In distraction osteogenesis, significantly elevated serum concentrations compared to baseline could be detected postoperatively for proMMP-1, MMP-9, TIMP-1, angiogenin and VEGF but not for proMMP-2, proMMP-3 or TIMP-2. In patients with rigid osteotomy healing, MMP-9, TIMP-1, TIMP-2, angiogenin and VEGF were significantly increased respectively. Comparison of both patient collectives revealed significantly higher increases of serum proMMP-1, VEGF and TIMP-1 in distraction patients during the lengthening period and significantly higher serum concentrations of TIMP-2 in late fracture healing period in osteotomy patients. Serum levels of MMP-13 were below the lowest standards, and therefore quantitative analysis was not possible. Bone regeneration in distraction osteogenesis and rigid osteotomy healing is accompanied by systemic increase of matrix-degrading and angiogenic factors in a certain time course and quantity. This might reflect biochemical regulation of local bone healing in the circulation. ProMMP-1, VEGF and TIMP-1 seem to be key regulatory factors during distraction osteogenesis.     

Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro.Human haematomas were collected from healthy patients undergoing surgery within 72 h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20% compression, 1 Hz) over 3 days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24 h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test.In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24 h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated.In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.     

Serum angiogenic activity: diagnostic relevance in renal cell carcinoma

OBJECTIVE: Angiogenesis is essential for tumor growth and progression. However, reported data on angiogenic parameters in patients with renal cell carcinoma are contradictory. The objective of this study was to use serum to compare the systemic angiogenic activity in patients with renal cell carcinoma and to determine if pathologic stage and grade correlated to this angiogenesis parameter. METHODS: Serum of 28 patients with a newly diagnosed renal cell carcinoma, 28 healthy volunteers and 9 patients with bladder carcinoma were used for this study. All sera were tested in a 72-hour endothelial cell proliferation assay. In addition the serum concentrations of the angiogenesis stimulators basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were determined using standard ELISA assays. RESULTS: The serum of renal cell carcinoma patients showed a median stimulation of human umbilical vein endothelial cells (HUVEC) of 89.79% (range 58.47-147.95%) and serum of healthy volunteers showed a median stimulation of 95.35% (range 74.64-141.77%) (p > 0.05). In contrast serum of patients with bladder carcinoma showed a median stimulation of 140.16% (range 64.82-200.16%) (p = 0.024). No correlations of the serum angiogenic activity and tumor stage or grade have been found in renal cell carcinoma patients. Furthermore, no correlations for serum bFGF and VEGF concentrations have been found. CONCLUSIONS: Serum angiogenic activity of patients with renal cell carcinoma did not differ significantly from healthy controls, while serum of patients with bladder carcinoma showed a significant increase in endothelial cell stimulation. Furthermore, bFGF and VEGF serum concentrations did not correlate to serum angiogenic activity in patients with renal cell carcinoma. Therefore, the determination of systemic angiogenic parameters, in case of renal cell carcinoma, might not lead to adequate data concerning prognosis or therapeutic effects.     

Elevated Serum Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Levels in Patients with Thymic Epithelial Neoplasms

Neovascularization, an essential event for the growth of solid tumors, is regulated by a number of angiogenic factors, among which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), are considered to exert potent angiogenic activity. In this study, we investigated whether serum VEGF and bFGF levels could be predictors of the development and extension of thymic epithelial neoplasms. The subjects of this study were 37 patients with thymoma, 6 with thymic carcinoma, and 23 healthy volunteers. Serum samples were collected before clinical treatment. Serum VEGF levels were significantly (P < 0.05) elevated in the patients with thymic carcinoma (1 080 ± 1 185 pg/ml) compared with those in the healthy volunteers (407 ± 589 pg/ml). Serum bFGF levels were also significantly (P < 0.05) elevated in the patients with thymic carcinoma (2 740 ± 631 pg/ml) compared with those in the healthy volunteers (1 728 ± 1 192 pg/ml). However, the serum VEGF and bFGF levels did not significantly differ between the patients with thymoma and the healthy volunteers. Serum VEGF and bFGF levels did not significantly differ according to the stage and pathological subtype of thymoma. Moreover, there was no correlation between the serum levels of VEGF and those of bFGF. Thus, while serum VEGF and bFGF levels may serve as markers for thymic epithelial tumors, it is unlikely that circulating VEGF and bFGF could be used as markers for assessing the progression of thymoma tumors. Received: November 10, 2000 / Accepted: May 15, 2001