Donor Specificity but Not Broadness of Sensitization Is Associated With Antibody-Mediated Rejection and Graft Loss in Renal Allograft Recipients.

Panel-reactive antibodies are widely regarded as an important immunological risk factor for rejection and graft loss. The broadness of sensitization against HLA is most appropriately measured by the "calculated population-reactive antibodies" (cPRA) value. In this study, we investigated whether cPRA represent an immunological risk in times of sensitive and accurate determination of pretransplantation donor-specific HLA antibodies (DSA). Five hundred twenty-seven consecutive transplantations were divided into four groups: cPRA 0% (n = 250), cPRA 1-50% (n = 129), cPRA 51-100% (n = 43), and DSA (n = 105). Patients without DSA were considered as normal risk and received standard immunosuppression without T cell-depleting induction. Patients with DSA received an enhanced induction therapy and maintenance immunosuppression. Surveillance biopsies were performed at 3 and 6 months. Median follow-up was 5.7 years. Among the three cPRA groups, there were no differences regarding the 1-year incidence of ABMR (p = 0.16) and TCMR (p = 0.75). The 5-year allograft survival rates were similar and around 87% (p = 0.28). The estimated glomerular filtration rate at last follow-up was 50-53 mL/min (p = 0.45). On multivariable Cox proportional hazard analysis, the strongest independent predictor for ABMR and (death-censored) graft survival was pretransplantation DSA. cPRA were not predictive for ABMR, TCMR, or (death-censored) graft survival. We conclude that with current DSA assignment, the broadness of sensitization measured by cPRA does not imply an immunological risk.

Pathology of resolving polyomavirus-associated nephropathy.

Control of polyomavirus BK (BKV) is achieved by reducing immunosuppression allowing an effective BKV-specific T-cell response. The morphology of resolving BKV-associated nephropathy (PyVAN) has not been systematically investigated. Ninety-nine surveillance biopsies of 35 patients with BKV viremia treated exclusively by immunosuppression reduction were scored according to Banff criteria and grouped relative to BKV viremia as pre-, increasing, decreasing and post-BKV viremia. Thirty-four of 35 patients (97%) cleared BKV viremia after a median of 9 months posttransplantation. The tubulitis score, extent of tubules with intraepithelial lymphocytes, and interstitial inflammation significantly increased from the time of increasing to decreasing viremia. Tubulointerstitial inflammation, to a lower extent, persisted after clearance. The number of SV40+ tubules correlated with the BKV load in plasma, but SV40 immunohistochemistry was frequently negative (60%). During decreasing viremia, 31% of PyVAN cases were plasma cell-rich and 40% showed tubular HLA-DR expression. Compared to baseline 1 month posttransplantation, allograft function remained stable or improved in 29/35 patients (83%) after a median follow-up of 48 months. Within 1 year after clearance of BKV viremia, clinical rejection occurred in 2/35 patients (6%). Our data suggest that resolving PyVAN is typically characterized by a self-limiting acute interstitial nephritis, morphologically indistinguishable from interstitial rejection.

Renal involvement in a patient with cobalamin A type (cblA) methylmalonic aciduria: a 42-year follow-up.

Chronic renal failure is a well-known long-term complication of methylmalonic aciduria (MMA-uria), occurring even under apparently optimal metabolic management. The onset of renal dysfunction seems to be dependent on the type of defect and vitamin B12-responsiveness. We report on a patient with a vitamin B12-responsive cobalamin A type (cblA) MMA-uria caused by a homozygous stop mutation (p.R145X) in the cobalamin A gene (MMAA). She was diagnosed with chronic kidney disease (CKD) stage III at the age of 12 years. Following re-evaluation, the patient received vitamin B12 (hydroxocobalamin) treatment, resulting in a significant decrease in the concentration of methylmalonic acid (MMA) in urine and plasma. Until age 29 years glomerular filtration rate remained stable probably due to hydroxocobalamin treatment slowing down progression to end-stage renal failure. Kidney biopsies showed non-specific manifestations of chronic interstitial inflammation. The patient received a renal transplant at age 35 years. Under continuous treatment with hydroxocobalamin there is no evidence of kidney damage due to MMA-uria until the last follow-up 6 years after transplantation. This case report illustrates (i) a long-term follow-up of a patient with MMA-uria due to cblA deficiency, (ii) the involvement of the kidney as a target organ and (iii) the importance of early and adequate vitamin B12 substitution in responsive patients. Further investigation will be necessary to prove the protective effect of hydroxocobalamin in the kidney in vitamin B12-responsive patients.

Detection of clinical and subclinical tubulo-interstitial inflammation by the urinary CXCL10 chemokine in a real-life setting.

Urinary CXCL10 is a promising noninvasive biomarker for tubulo-interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real-life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2-3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo-interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1-3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10-guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.

Is monitoring of FOXP3 Treg cells in renal transplants during acute cellular rejection episodes useful?

BACKGROUND: The FOXP3 (forkhead Box p3) transcription factor is a marker for T regulatory cells (Treg). During cellular immune responses, Treg are expected to increase in number to ultimately control and limit this response. In renal transplants massive infiltration by T cells is often seen during rejection crises. This prompted us to examine changes in the numbers of FOXP3 positive T cells accompanying acute cellular rejection events. METHODS: A total of 32 transplant biopsies from 23 patients were studied retrospectively, these 16 protocol biopsies and 16 biopsies taken during rejection episodes included 9 serial pairs (protocol-rejection). To quantify FOXP3 positive T cells, frozen sections were double immunostained with anti-CD3 and anti-FOXP3 antibodies. Areas revealing T cell infiltrates were measured morphometrically and the number of FOXP3 positive cells per 1,000 µm2 of CD3 positive cells was taken as an FOXP3 index. RESULTS: This index was 0.46 (median, range 0.00-1.00) in the 16 protocol biopsies and 0.48 (median, range 0.16-2.31) in rejection episode biopsies. The highest values were seen during rejection crises, exceeding 1.00 in 6/16 biopsies, whereas no protocol biopsies had values greater than 1.00 (0/16) (difference significant p<0.02). In serial biopsies no consistent behavior was observed; the FOXP3 index remained unchanged, fell slightly or rose to a maximum of 13 fold. Expression levels of FOXP3 could vary within weeks. No correlations were found between donor type, initial therapy, therapy at biopsy, serum creatinine at the time of biopsy, at 3 months or 1 year later, and any of the morphometric parameters (CD3 and FOXP3) studied. CONCLUSIONS: During rejection of renal allografts the fraction of FOXP3+ Treg cells within the infiltrating T-cell population can increase transiently. This phenomenon was not consistently seen in acute cellular rejection and the information does not appear to be of value for individual patient management in such cases.

Reducing immunosuppression preserves allograft function in presumptive and definitive polyomavirus-associated nephropathy.

Early detection of polyomavirus BK (BKV) viremia and reduction of immunosuppression is recommended for preventing polyomavirus-associated nephropathy (PyVAN), but systematic histological evaluations were not performed in previous studies. We routinely screen for decoy cells and, if positive, measure plasma BKV-loads. In a cohort of 203 consecutive renal transplantations performed from 2005-2008, 38 patients (19%) developed BKV-viremia and were treated with reduction of immunosuppression. Based on subsequent allograft biopsy results and peak BKV-viremia, patients were assigned to three groups: (i) definitive PyVAN (n = 13), (ii) presumptive PyVAN defined by plasma BKV-loads of ≥ 4 log(10) copies/ml (n = 17) and (iii) low BKV-viremia (n = 8). Clearance of BKV-viremia was achieved in 35/38 patients (92%) and subsequent clinical rejection occurred in 3/35 patients (8.6%), both without any difference among the groups. Patients with definitive PyVAN had higher peak plasma BKV-loads and required longer time for clearance (8.8 vs. 4.6 vs. 2.9 months; p = 0.001). However, allograft function remained stable from baseline to last follow-up at 34 months (range 18-60) in all three groups with median serum creatinine of 1.6 mg/dl, 1.6 mg/dl and 1.3 mg/dl, respectively. We conclude that screening for BKV-replication and reduction of immunosuppression is an effective strategy to preserve medium-term allograft function even in patients developing definitive PyVAN.

Efficacy of induction therapy with ATG and intravenous immunoglobulins in patients with low-level donor-specific HLA-antibodies.

Low-level donor-specific HLA-antibodies (HLA-DSA) (i.e. detectable by single-antigen flow beads, but negative by complement-dependent cytotoxicity crossmatch) represent a risk factor for early allograft rejection. The short-term efficacy of an induction regimen consisting of polyclonal anti-T-lymphocyte globulin (ATG) and intravenous immunoglobulins (IvIg) in patients with low-level HLA-DSA is unknown. In this study, we compared 67 patients with low-level HLA-DSA not having received ATG/IvIg induction (historic control) with 37 patients, who received ATG/IvIg induction. The two groups were equal regarding retransplants, HLA-matches, number and class of HLA-DSA. The overall incidence of clinical/subclinical antibody-mediated rejection (AMR) was lower in the ATG/IvIg than in the historic control group (38% vs. 55%; p = 0.03). This was driven by a significantly lower rate of clinical AMR (11% vs. 46%; p = 0.0002). Clinical T-cell-mediated rejection (TCR) was significantly lower in the ATG/IvIg than in the historic control group (0% vs. 50%; p < 0.0001). Within the first year, allograft loss due to AMR occurred in 7.5% in the historic control and in 0% in the ATG/IvIg group. We conclude that in patients with low-level HLA-DSA, ATG/IvIg induction significantly reduces TCR and the severity of AMR, but the high rate of subclinical AMR suggests an insufficient control of the humoral immune response.

Individual effect of renin-angiotensin system inhibition and corticosteroid therapy in IgA glomerulonephritis: results of a pilot study.

BACKGROUND: The impact of different therapy modalities on the outcome of Immunoglobulin A glomerulonephritis (IgAGN) in individual patient is not clear. We present preliminary results from the sequential application of renin-angiotensin system (RAS) inhibition and corticosteroids to discriminate the individual effect of both therapies. METHODS: Regardless of the degree of proteinuria, renal function and histological grading, patients with biopsy-proven IgAGN were treated with a standardized protocol. RAS inhibition was performed for 3 months. Thereafter, immunosuppressive therapy with prednisone (0.5 mg/kg body weight) on alternate days for 6 months was started. The primary endpoint was a maximal reduction of proteinuria (spot urine protein/ creatinine ratio (uPCR)), by RAS inhibition and by the combination of RAS inhibition and steroids. RESULTS: 10 patients were treated according to the protocol. During a median follow-up of 18 months, uPCR decreased from initial 230 mg/mmol (2 g/g) (median, interquartile range (IQR) 146 - 396) to 154 mg/mmol (1.4 g/g) (IQR 88 - 190) at 3 months during the RAS inhibition period (33% reduction, p = 0.01) and further to 31 mg/mmol (0.3 g/g) (IQR 21 - 71) until end of the steroid period at 9 months (80% reduction compared to uPCR at 3 month, p < 0.001). At the last F/U, uPCR (median) remained stable at 41 mg/mmol (0.4 g/g). The estimated glomerular filtration rate was stable during the whole observation period. CONCLUSIONS: Sequential RAS inhibition and steroid treatment leads to a continuous decrease in proteinuria, beyond the decrease produced by isolated RAS inhibition. Our data suggest independent effects of both, RAS inhibition and steroids, on the reduction of proteinuria in a small, non selected group of patients with IgAGN. The hypothesis that patients with IgAGN, regardless of the degree of proteinuria, renal function and histological grading, may benefit from combination therapy with maximal RAS inhibition and low dose corticosteroids now has to be confirmed in a randomized study.

Acute renal failure associated with bilateral enlargement of the kidneys: a rare manifestation of acute lymphoblastic leukemia (ALL).

We report a 6-year-old patient who presented with acute renal failure resolving after vigorous intravenous hydration. Renal biopsy was taken because of unexplained enlargement of both kidneys. Histological workup showed infiltration by lymphoblasts while blood counts showed a normal differential. Subsequent bone marrow aspiration revealed 34% lymphoblasts of T-lineage origin, leading to the diagnosis of T-ALL. This case underlines that malignant hematologic infiltration should be considered in patients presenting with unexplained renal failure and enlarged kidneys.

Anti-angiogenic cues from vascular basement membrane collagen.

Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important antiangiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the alpha1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the alpha1beta1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.

A Comparison of Two Types of Rabbit Antithymocyte Globulin Induction Therapy in Immunological High-Risk Kidney Recipients: A Prospective Randomized Control Study.

BACKGROUND: Induction treatment with rabbit polyclonal antithymocyte globulins (ATGs) is frequent used in kidney transplant recipients with donorspecific HLA antibodies and shows acceptable outcomes. The two commonly used ATGs, Thymoglobulin and ATG-F have slightly different antigen profile and antibody concentrations. The two compounds have never been directly compared in a prospective trial in immunological high-risk recipients. Therefore we performed a prospective randomized controlled study comparing the two compounds in immunological high-risk kidney recipients in terms of safety and efficacy. METHODS: Immunological high-risk kidney recipients, defined as the presence of HLA DSA but negative CDC-B and T-cell crossmatches were randomized 1:1 to receive ATG-F or Thymoglobulin. Maintenance immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil and steroids. RESULTS: The per-protocol analysis included 35 patients. There was no immediate infusion reaction observed with both compounds. No PTLD or malignancy occurred during the follow-up in both groups. The incidence of viral and bacterial infections was similar in both groups (p = 0.62). The cumulative incidence of clinical and subclinical antibody mediated allograft rejection as well as T-cell mediated allograft rejection during the first year between ATG-F and Thymoglobulin was similar (35% versus 19%; p = 0.30 and 11% versus 18%; 0.54 respectively). The two-year graft function was similar with a median eGFR of 56 ml/min/1.73m2 (range 21-128) (ATG-F-group) and 51 ml/min/1.73m2 (range 22-132) (Thymo-group) (p = 0.69). CONCLUSION: We found no significant differences between the compared study drugs for induction treatment in immunological high-risk patients regarding safety and efficacy during follow-up with good allograft function at 2 years after transplantation.

In vitro interactions of endometrial stromal and epithelial cells in Matrigel: reorganization of the extracellular matrix.

Fragments of human endometrial glands and dispersed endometrial stromal cells were cultured together in a thick layer of reconstituted basement membrane (Matrigel). Epithelial cells kept their glandular morphology whereas stromal cells grew into round clusters of mainly fusiform cells. Transmission electron micrographs showed collagen fibers between stromal cells as well as in surrounding extracellular matrix after 2.5 weeks. A well-defined basement membrane was found when epithelial and stromal cells were in close proximity to each other. Beneath the lamina densa there was a loose network of collagen fibers or a dense fibrillar network arranged parallel to the cell layers. Epithelial cells showed hemidesmosomes at their basal surface where they were close to stromal cells.

Basement membrane induced differentiation of HEC-1B(L) endometrial adenocarcinoma cells affects both morphology and gene expression.

In vitro studies of endometrial carcinogenesis have been hampered by dedifferentiation of the cells in culture. Using the endometrial carcinoma cell line HEC-1B(L), we aimed to establish and characterize culture conditions that preserve a more differentiated state of the tumor cells. HEC-1B(L) cells grown in a serum-free defined medium on plastic (PL/SFDM) on top of a reconstituted basement membrane (Matrigel, MG/SFDM) or in a thick layer of Matrigel showed pronounced morphological differentiation as compared with HEC-1B(L) cells cultured on plastic in a medium containing serum (PL/10% FCS). Features of differentiation included cuboidal to columnar cell shape and an increase of rough endoplastic reticulum in Matrigel cultures. Gene expression of HEC-1B(L) cells was studied by metabolic [35S]methionine labeling and SDS-gel electrophoresis. HEC-1B(L) cells cultured in the presence of Matrigel showed two additional secretory proteins approximately 31 kD and 77 kD in size. rt-PCR was used to screen cell cultures for the presence of estrogen receptor, progesterone receptor, and lactoferrin-mRNA, genes typically expressed by normal endometrial epithelium. We found no expression of the estrogen receptor or progesterone receptor. Lactoferrin-mRNA was present under all culture conditions tested. Our results suggest a regulatory role of the extracellular matrix for the differentiation of the HEC-1B(L) cell line.

Laminin mediates basement membrane induced differentiation of HEC 1B endometrial adenocarcinoma cells.

In vitro studies on endometrial carcinogenesis have been hampered by limited differentiation of the cells in culture. Using the endometrial carcinoma cell lines HEC 1B and its subclone HEC 1B(L), we established and characterized cell culture conditions that preserve a more differentiated state of the tumor cells. Randomly seeded HEC 1B(L) cells, if grown in a serum-free defined medium on top of a reconstituted basement membrane (Matrigel), within a few hours assembled themselves to web-like structures. In a thick layer of Matrigel, they showed an even more pronounced morphological differentiation. Functionally, two additional secretory proteins, about 31 and 77 kDa in size, became apparent as a response to matrigel. To further investigate the regulatory role of the extracellular matrix in the process of in vitro differentiation of endometrial adenocarcinoma cells, we addressed two specific problems. First, we investigated if the capacity of in vitro differentiation is a specific feature of HEC 1B(L) cells or if it is common to all endometrial adenocarcinoma cells. Second, we tried to identify the Matrigel component(s) responsible for in vitro differentiation. The assembly of HEC 1B and HEC 1B(L) cells into spatially organized web-like structures and the expression of the 77 kDa protein were thereby used as an assay. All endometrial adenocarcinoma cell lines tested to a variable degree formed web-like structures on Matrigel. Although the pattern of de novo synthesized secretory proteins changed as a response to Matrigel, only HEC 1A, HEC 1B, HEC 1B(L), and Ishikawa cells responded to culture on Matrigel by an increased expression of the 77 kDa protein. Functionally, polyclonal anti-laminin antibodies, but not anti-collagen type IV antibodies, disrupted formation of web-like structures by HEC 1B cells. The laminin-specific peptides YIGSR and SIKVAV but none of the RGD-peptides RGDS, GRGDSP, or GRADSP affected the three-dimensional assembly of these cells in vitro. Both anti-laminin antibodies and laminin-specific peptides suppressed Matrigel-induced formation of the 77-kDa secretory protein by HEC 1B cells. These findings suggest the involvement of laminin in the in vitro differentiation of the HEC 1B endometrial adenocarcinoma cell line. In a mechanistic view, laminin appears to play a crucial role in the regulation of this in vitro differentiation process.

Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin.

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.

Distinct antitumor properties of a type IV collagen domain derived from basement membrane.

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth.

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.