Endoglin Deficiency Research Articles

Deficiency in hereditary hemorrhagic telangiectasia-associated Endoglin elicits hypoxia-driven heart failure in zebrafish.

Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-β (TGF-β) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model.

Endoglin deficiency impairs VEGFR2 but not FGFR1 or TIE2 activation and alters VEGF-mediated cellular responses in human primary endothelial cells

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease characterized by vascular dysplasia. Mutations of the endoglin (ENG) gene that encodes a co-receptor of the transforming growth factor β1 signaling pathway cause type I HHT. ENG is primarily expressed in endothelial cells (ECs), but its interaction with other key angiogenic pathways to control angiogenesis has not been well addressed. The aim of this study is to investigate ENG interplay with VEGFR2, FGFR1 and TIE2 in primary human ECs. ENG was knocked-down with siRNA in human umbilical vein ECs (HUVECs) and human lung microvascular ECs (HMVEC-L). Gene expression was measured by RT-qPCR and Western blotting. Cell signaling pathway activation was analyzed by detecting phosphor-ERK and phosphor-AKT levels. Cell migration and apoptosis were assessed using the Boyden chamber assay and the CCK-8 Kit, respectively. Loss of ENG in HUVECs led to significantly reduced expression of VEGFR2 but not TIE2 or FGFR1, which was also confirmed in HMVEC-L. HUVECs lacking ENG had significantly lower levels of active Rac1 and a substantial reduction of the transcription factor Sp1, an activator of VEGFR2 transcription, in nuclei. Furthermore, VEGF- but not bFGF- or angiopoietin-1-induced phosphor-ERK and phosphor-AKT were suppressed in ENG deficient HUVECs. Functional analysis revealed that ENG knockdown inhibited cell migratory but enhanced anti-apoptotic activity induced by VEGF. In contrast, bFGF, angiopoietin-1 and -2 induced HUVEC migration and anti-apoptotic activities were not affected by ENG knockdown. In conclusion, ENG deficiency alters the VEGF/VEGFR2 pathway, which may play a role in HHT pathogenesis.

Endoglin in human liver disease and murine models of liver fibrosis-A protective factor against liver fibrosis.

Background & AimsLiver fibrosis is the outcome of chronic liver injury. Transforming growth factor‐β (TGF‐β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF‐β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC‐specific endoglin deletion.MethodsEng expression was measured in human and murine samples of liver injury. After generating GFAPCre(+)EngΔ HSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Engflox/flox HSCs were infected with Cre‐expressing virus to deplete Endoglin and fibrogenic responses were analysed.ResultsEndoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L‐Eng splice variant. Comparing GFAPC re(−)Engf/f to GFAPC re(+)EngΔ HSC mice in toxic liver injury, livers of GFAPC re(+)EngΔ HSC mice showed 39.9% (P < .01) higher Hydroxyproline content compared to GFAPC re(−)Engf/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P < .05) more Collagen deposition in livers of GFAPC re(+)EngΔ HSC mice. Similar results were obtained in mice subjected to cholestatic injury.ConclusionEndoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α‐SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF‐β signalling.

Modulation of endoglin expression in islets of langerhans by VEGF reveals a novel regulator of islet endothelial cell function.

BackgroundEndoglin/CD105 is an auxiliary receptor for transforming growth factor-β with established roles in vascular remodelling. It has recently been shown that heterozygous endoglin deficiency in mice decreases insulin secretion in an animal model of obesity, highlighting a potential role for endoglin in the regulation of islet function. We have previously identified two different populations of endoglin expressing cells in human and mouse islets which are: (i) endothelial cells (ECs) and (ii) islet mesenchymal stromal cells. The contribution of islet EC endoglin expression to islet development and sensitivity to VEGF is unknown and is the focus of this study.ResultsIn vitro culture of mouse islets with VEGF164 for 48 h increased endoglin mRNA levels above untreated controls but VEGF did not modulate VEGFR2, CD31 or CD34 mRNA expression or islet viability. Removal of EC-endoglin expression in vivo reduced islet EC area but had no apparent effect on islet size or architecture.ConclusionEC-specific endoglin expression in islets is sensitive to VEGF and plays partial roles in driving islet vascular development, however such regulation appears to be distinct to mechanisms required to modulate islet viability and size.

BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses

Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial role in mechanotransduction and cell-cell contact. Identification of BMP9-dependent nuclear localization of YAP1 in response to endoglin expression suggests a mechanism of crosstalk between the two pathways. Suppression of endoglin and YAP1 alters BMP9-dependent expression of YAP1 target genes CCN1 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF) as well as the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1). These results suggest a coordinate effect of endoglin deficiency on cell matrix remodeling and local inflammatory responses. Identification of a direct link between the Hippo pathway and endoglin may reveal novel mechanisms in the etiology of HHT.

Endoglin deficiency impairs stroke recovery.

Endoglin deficiency causes hereditary hemorrhagic telangiectasia-1 and impairs myocardial repair. Pulmonary arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia-1 are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that endoglin deficiency impairs stroke recovery. Eng heterozygous (Eng+/-) and wild-type mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Pial collateral vessels were quantified before pMCAO. Infarct/atrophic volume, vascular density, and macrophages were quantified in various days after pMCAO, and behavioral function was assessed using corner and adhesive removal tests on days 3, 15, 30, and 60 after pMCAO. The association between ENG 207G>A polymorphism and brain arteriovenous malformation rupture and surgery outcome was analyzed using logistic regression analysis in 256 ruptured and 157 unruptured patients. After pMCAO, Eng+/- mice showed larger infarct/atrophic volumes at all time points (P<0.05) and showed worse behavior performance (P<0.05) at 15, 30, and 60 days when compared with wild-type mice. Eng+/- mice had fewer macrophages on day 3 (P=0.009) and more macrophages on day 60 (P=0.02) in the peri-infarct region. Although Eng+/- and wild-type mice had similar numbers of pial collateral vessels before pMCAO, Eng+/- mice had lower vascular density in the peri-infarct region (P=0.05) on day 60 after pMCAO. In humans, ENG 207A allele has been associated with worse outcomes after arteriovenous malformation rupture or surgery of patients with unruptured arteriovenous malformation. Endoglin deficiency impairs brain injury recovery. Reduced angiogenesis, impaired macrophage homing, and delayed inflammation resolution could be the underlying mechanism.

Mouse bone marrow-derived endothelial progenitor cells do not restore radiation-induced microvascular damage.

Background. Radiotherapy is commonly used to treat breast and thoracic cancers but it also causes delayed microvascular damage and increases the risk of cardiac mortality. Endothelial cell proliferation and revascularization are crucial to restore microvasculature damage and maintain function of the irradiated heart. We have therefore examined the potential of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) for restoration of radiation-induced microvascular damage. Material & Methods. 16 Gy was delivered to the heart of adult C57BL/6 mice. Mice were injected with BM-derived EPCs, obtained from Eng+/+ or Eng+/− mice, 16 weeks and 28 weeks after irradiation. Morphological damage was evaluated at 40 weeks in transplanted mice, relative to radiation only and age-matched controls. Results. Cardiac irradiation decreased microvascular density and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor). Microvascular damage was not diminished by treatment with BM-derived EPCs. However, BM-derived EPCs from both Eng+/+ and Eng+/− mice diminished radiation-induced collagen deposition. Conclusion. Treatment with BM-derived EPCs did not restore radiation-induced microvascular damage but it did inhibit fibrosis. Endoglin deficiency did not impair this process.

TGF-β & BMP receptors endoglin and ALK1: overview of their functional role and status as antiangiogenic targets.

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF-β superfamily, TGF-β1, TGF-β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell-cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1-mediated cells signaling.

136 ENDOGLIN DEFICIENCY IN HEPATIC STELLATE CELLS HAS A DIFFERENTIAL EFFECT ON LIVER FIBROSIS AND TGF-b SIGNALLING IN TWO EXPERIMENTAL MODELS OF MURINE LIVER FIBROSIS

Background: Hepatic stellate cells (HSCs) are the major source for extracellular matrix (ECM) production in liver fibrosis. Endoglin (ENG) is a type III auxiliary receptor for TGF-b that is expressed on proliferating endothelial cells and HSCs. TGF-b is the most profibrotic cytokine expressed in response to liver injury. This study analyzes the role of ENG and TGF-b signalling in two models of experimental liver fibrosis by cell line specific Endoglin deletion in HSCs. Methods: Using the Cre-LoxP genetic recombination system we created HSC specific ENG mice by crossing GFAP to ENG mice. Mice were subjected to liver injury by CCl4 treatment (8 weeks) or bile duct ligation (BDL) (21 days). Liver fibrosis was analyzed by hydroxyproline measurement and Sirius red staining for collagen fibers. For in vitro analysis HSCs from ENG mice were isolated and treated with adenoviral CMV-RSV expressing vector. TGF-b signalling was analyzed by western blots and qPCR. Results: In response to CCl4 treatment livers of GFAP Eng mice (n =8) showed 18.2% less hydroxyproline content compared to GFAPEng /f litter mates reflecting less severe fibrosis. Sirius red stainings underlined these findings. Interestingly BDL inflicted injury showed contrasting results. Compared to GFAPEng /f mice GFAPEng litter mates (n = 8) showed 193.3% (p =0.02) more ECM deposition. Western blot analysis of TGF-b signalling in vitro using adenoviral Cre treated ENG primary HSCs demonstrated a differential effect of ENG on TGF-b signalling. In the absence of ENG activation of Smad1/5/8 was downregulated. Moreover, the phosphorylation of ERK1/2 was decreased and the expression of a-smooth muscle actin and connective tissue growth factor was downregulated. Conclusion: Endoglin deficiency in hepatic stellate cells has a differential effect on liver fibrosis depending on the cause of injury. Hepatocyte necrosis due to CCl4 treatment leads to strong TGF-b expression Smad signal transduction. Endoglin deficiency leads to amelioration of liver fibrosis, underlined by in vitro results. In contrast cholestatic liver injury is aggravated by endoglin deficiency in HSCs, reflecting a complex interplay of cholestasis, inflammation and toxic injury. Endoglin obviously modulates TGF-b signalling in this periportal-centered injury differentially, leading to significantly more fibrotic changes. 137 CXCL1 AND CXCL10 SECRETION BY FIBROCYSTIN-DEFECTIVE CHOLANGIOCYTES RECRUITS PERIBILIARY FIBROCYTES IN CONGENITAL HEPATIC FIBROSIS L. Fabris, L. Locatelli, D. Vigano, M. De Matteis, R. Fiorotto, M. Cadamuro, C. Spirli, M. Strazzabosco. Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA E-mail: luca.fabris@unipd.it

Abstract TMP69: Endoglin Deficiency Exacerbates Ischemic Brain Injury

Background and Objective: Endoglin (Eng) deficiency causes hereditary hemorrhagic telangiectasia (HHT) and impairs myocardial repair. Pulmonary arteriovenous malformations in HHT patients are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that Eng deficiency exacerbates ischemic brain injury. Methods: Eng heterozygous ( Eng +/- ) mice and wild type (WT) mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Infarct volume and CD68 + cells were quantified 3 days and vascular density was determined 60 days after pMCAO. Behavior was assessed by corner test and adhesive removal test at 3, 15, 30 and 60 days after pMCAO. Matrix metalloproteinase 9 (Mmp9) and Notch1 expression in bone marrow (BM)-derived macrophages from Eng +/- and WT were analyzed using real-time RT-PCR. Results: Eng +/- mice had a larger Infarct volume than WT mice (22±6% of the affected hemisphere vs. 16±6%, p=0.04). Eng +/- mice had longer adhesive-removal time (p&lt;0.05) and more frequent turning to the lesion side than WT mice at 15, 30 and 60 days (p&lt;0.05) after pMCAO. Both groups had similar numbers of CD68 + cells in the peri-infarct area at 3 days after pMCAO (370±80 vs 338±44 cells/mm 2 , p=0.37), but Eng +/- mice had lower peri-infarct vessel density (417±69 vs 490±52 vessels/mm 2 , p=0.05) at 60 days after pMCAO. Up-regulation of Mmp9 and Notch1 expression in response to VEGF was attenuated in Eng +/- BM-derived macrophages. Conclusions: Endoglin deficiency exacerbated brain injury and behavior dysfunction in mice after pMCAO and was associated with reduced angiogenesis. Although macrophage homing was not affected, reduced expression of two angiogenic-related genes, Mmp9 and Notch1 , by Eng +/- BM-derived macrophages suggests a potential role of these cells in recovery from an ischemic injury.

Heterozygous deficiency of endoglin decreases insulin and hepatic triglyceride levels during high fat diet.

Endoglin is a transmembrane auxiliary receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells. It plays a wide range of physiological roles but its importance on energy balance or insulin sensitivity has been unexplored. Endoglin deficient mice die during midgestation due to cardiovascular defects. Here we report for first time that heterozygous endoglin deficiency in mice decreases high fat diet-induced hepatic triglyceride content and insulin levels. Importantly, these effects are independent of changes in body weight or adiposity. At molecular level, we failed to detect relevant changes in the insulin signalling pathway at basal levels in liver, muscle or adipose tissues that could explain the insulin-dependent effect. However, we found decreased triglyceride content in the liver of endoglin heterozygous mice fed a high fat diet in comparison to their wild type littermates. Overall, our findings indicate that endoglin is a potentially important physiological mediator of insulin levels and hepatic lipid metabolism.

Endoglin Deficiency in Bone Marrow is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse After Vascular Endothelial Growth Factor Stimulation

Bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced brain angiogenic foci, and VEGF induces cerebrovascular dysplasia in adult endoglin heterozygous (Eng(+/-)) mice. We hypothesized that Eng(+/-) BMDCs cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. BM transplantation was performed using adult wild-type (WT) and Eng(+/-) mice as donors/recipients. An adeno-associated viral vector expressing VEGF was injected into the basal ganglia 4 weeks after transplantation. Vascular density, dysplasia index (vessels >15 µm/100 vessels), and BMDCs in the angiogenic foci were analyzed. The dysplasia index of WT/Eng(+/-) BM mice was higher than WT/WT BM mice (P<0.001) and was similar to Eng(+/-)/Eng(+/-) BM mice (P=0.2). Dysplasia in Eng(+/-) mice was partially rescued by WT BM (P<0.001). WT/WT BM and WT/Eng(+/-) BM mice had similar numbers of BMDCs in the angiogenic foci (P=0.4), most of which were CD68(+). Eng(+/-) monocytes/macrophages expressed less matrix metalloproteinase-9 and Notch1. Endoglin-deficient BMDCs are sufficient for VEGF to induce vascular dysplasia in the adult mouse brain. Our data support a previously unrecognized role of BM in the development of cerebrovascular malformations.

BMP9 regulates endoglin-dependent chemokine responses in endothelial cells

AbstractBMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.

Abstract 3153: Endoglin Deficiency in Bone Marrow is Sufficient to Cause Vascular Dysplasia in the Adult Mouse Brain After VEGF Stimulation

Background and Objective: We have previously demonstrated that VEGF overexpression in the brain of adult Eng+/- mice causes vascular abnormalities. We also found that bone marrow-derived cells (BMDCs) home to vascular endothelial growth factor (VEGF)-induced angiogenic foci in the adult mouse brain and contribute to angiogenesis. Impaired angiogenesis in infarcted myocardium of Eng+/- mice was rescued using treatment with healthy human BMDCs but not with BMDCs from Hereditary Hemorrhagic Telangiectasia (HHT1) patients carrying ENG mutations. We hypothesized that ENG-haploinsufficiency in BMDCs can cause cerebrovascular dysplasia in the adult mouse after VEGF stimulation. Methods: WT or Eng+/- BM (2×106 cells) were transplanted to lethally irradiated C57BL/6 recipient mice (8-10 weeks old) via tail vein injection. After 4 weeks of BM transplantation, adeno-associated viral vector expressing VEGF (AAV-VEGF, 2×109 genome copies) was stereotactically injected into the basal ganglia. Brain sections were collected at 4 weeks after virus injection. The vascular density (vessels per 20× objective field) and dysplasia index (number of vessels &gt;15µm in diameter per 200 vessels) were quantified on lectin-stained brain sections. Results: To examine the angiogenic response to VEGF, the vascular density around the AAV-VEGF injection site was analyzed. The mean vascular density increased in all mice with VEGF stimulation. Mice transplanted with Eng+/- BM showed less vascular density compared to mice transplanted with WT BM. The mean vascular densities were167 ± 21 (Eng+/-BM, n=4) and 202 ± 15 (WT BM, n=5) (P&lt;0.01). Further, mice with WT BM had angiogenesis with normal vascular morphology. In contrast, mice with Eng+/- BM had markedly enlarged and dysmorphic vessels. To assess the degree of the dysplastic response, dysplasia index was analyzed. Mice with Eng+/- BM had several fold-higher dysplasia index than mice with WT BM: 2.6 ± 1.4 (Eng+/- BM) versus 0.7 ± 0.4 (WT BM) (P&lt;0.01). Conclusions: In the adult mouse after VEGF stimulation, ENG-deficiency in BM (1) caused a 17% reduction in brain angiogenic response, and (2) increased cerebrovascular dysplasia, compared to control animals. These data support the possibility that cerebrovascular malformations resulting from VEGF stimulation with ENG-haploinsufficiency is attributable to impaired cell-autonomous monocyte/macrophage function, in contrast to the view that ENG dysfunction is primarily an endothelial phenomenon.

13-P035 Endoglin deficiency leads to arteriovenous malformations during angiogenesis

13-P035 Endoglin deficiency leads to arteriovenous malformations during angiogenesis

Genistein Induces Phenotypic Reversion of Endoglin Deficiency in Human Prostate Cancer Cells

Genistein has been shown to inhibit human prostate cancer (PCa) cell motility. Endoglin has been identified as an important suppressor of PCa cell motility, and its expression is lost during PCa progression. It is therefore important to determine whether endoglin loss affects genistein's efficacy and, if so, by what mechanism. In the current study, genistein was shown to induce reversion of endoglin-deficient cells to a low motility, endoglin-replete phenotype. Because endoglin suppresses PCa cell motility in an activin-like kinase receptor-2 (ALK2)- and Smad1-dependent manner, we sought to determine whether genistein was activating the ALK2-Smad1 pathway. Although treatment with genistein or overexpression of Smad1 or ALK2 all increased Smad1-responsive promoter activity and decreased cell motility, genistein's efficacy was abrogated by either Smad1 or ALK2 knockdown. Furthermore, transfection of cells with a kinase dead mutant of ALK2 abrogated genistein's efficacy. Together, these findings demonstrate that genistein therapeutically induces reversion to a low-motility phenotype in aggressive endoglin-deficient PCa cells. It does so by activating ALK2-Smad1 endoglin-associated signaling. These findings support the notion that individuals with low endoglin-expressing PCa will benefit from genistein treatment.