Endoglin Mouse

What is endoglin and what are its primary functions in mouse biology?

Endoglin (CD105) is a transmembrane type III receptor for TGF-beta superfamily ligands that plays critical roles in smooth muscle differentiation, angiogenesis, and neovascularization. It functions by associating with receptors such as TGF-beta RII, Activin RIIA or RIIB, BMPR-IA/ALK-3, or BMPR-IB/ALK6, enhancing binding of ligands including Activin A, BMP-2, -7, -9, TGF-beta 1, and TGF-beta 3 . In mice, endoglin has been identified as a functional marker that defines long-term repopulating (LTR) hematopoietic stem cells within bone marrow side-population cells . Importantly, endoglin can either enhance or inhibit signaling through its receptor complexes, suggesting a context-dependent regulatory role .

Which splice variants of endoglin exist in mice and how do they differ in expression patterns?

Mice express two main endoglin splice variants:

• L-Endoglin (long form): This is the predominant isoform expressed in most mouse tissues and isolated liver cells including Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) .

• S-Endoglin (short form): This variant is also present in significant levels in various tissues and liver cells .

Expression analysis shows differential regulation of these variants in disease states. While L-Eng expression increases in both acute liver failure and chronic liver injuries, S-Eng levels are significantly elevated only in acute liver failure patients but not in chronic conditions like NASH or HCV infection .

How does endoglin expression change during liver injury in mouse models?

In murine models of liver injury, both L-Endoglin and S-Endoglin show significant upregulation compared to control animals. After bile duct ligation (BDL), a notable pattern emerges:

• S-Endoglin shows more pronounced upregulation early after injury

• By the second and third week after BDL, both isoforms become highly expressed compared to controls

Similar expression patterns were observed in CCl₄-treated animals, confirming that endoglin upregulation is a consistent response to various forms of liver injury .

What phenotypes do endoglin heterozygous (Eng+/-) mice exhibit and how do they serve as disease models?

Endoglin heterozygous (Eng+/-) mice serve as an established model for hereditary hemorrhagic telangiectasia (HHT), a vascular disorder characterized by arteriovenous malformations. These mice exhibit several key phenotypes:

• Reduced endothelial NO synthase (eNOS) expression with impaired activity

• Specific upregulation of cyclooxygenase-2 (COX-2) in vascular endothelium

• Increased urinary excretion of prostaglandin E₂

• Abnormal responses to COX-2 inhibition (transient increase in arterial pressure not observed in wild-type mice)

• Increased susceptibility to abnormal microvessel formation when exposed to VEGF overexpression, including enlarged vessels, clustering, twisting, or spiral formations

These phenotypes make Eng+/- mice valuable for studying vascular malformation development and for testing therapeutic interventions targeting the TGF-β signaling pathway.

How does endoglin deletion in hepatic stellate cells affect liver fibrosis progression?

Targeted deletion of endoglin in hepatic stellate cells (HSCs) using GFAP-Cre recombinase (GFAP Cre(+)Eng ΔHSC mice) has significant impacts on liver fibrosis progression:

• In toxic liver injury models, GFAP Cre(+)Eng ΔHSC mice exhibited:

  • 39.9% higher hydroxyproline content (p<0.01) compared to control littermates

  • 58.8% more collagen deposition (p<0.05) as assessed by Sirius Red staining

• Similar results were observed in cholestatic injury models, indicating endoglin's protective role against excessive fibrosis

These findings suggest that endoglin expression in HSCs functions as a negative regulator of fibrogenesis, and its absence exacerbates the fibrotic response to liver injury.

What is the relationship between endoglin and VEGF-induced angiogenesis in mouse models?

VEGF overexpression in Eng+/- mice leads to distinct vascular abnormalities not observed in wild-type (Eng+/+) counterparts:

• While VEGF overexpression increases microvessel count for up to 4 weeks in both Eng+/+ and Eng+/- groups (185±14 vs. 201±10 microvessels/mm²), the morphology of these vessels differs markedly

• Confocal microscopic examination revealed grossly abnormal microvessels in eight of nine Eng+/- mouse brains compared with zero of nine in Eng+/+ mice (p<0.05)

• Abnormal microvessels featured distinctive characteristics: enlargement, clustering, twisting, or spiral formations

• VEGF receptor Flk-1 and TGF-β receptor 1 (TβR1) expression were reduced in Eng+/- mouse brains compared to controls

These findings demonstrate that endoglin haploinsufficiency creates a permissive environment for VEGF-induced vascular malformations, potentially through altered receptor expression and signaling pathways.

What are the most effective methods for quantifying endoglin protein in mouse samples?

For accurate quantification of mouse endoglin protein, the Quantikine Mouse Endoglin/CD105 ELISA kit provides a reliable methodological approach with the following performance characteristics:

Technical Specifications:

• Assay duration: 4.5 hours

• Sample types: Cell culture supernatants, cell lysates, serum, EDTA plasma, heparin plasma, urine

• Precision:

  • Intra-assay CV%: 2.7-5.2%

  • Inter-assay CV%: 4.9-8.7%

Recovery Rates by Sample Type:

This assay provides consistent results across different sample types with excellent recovery rates, making it suitable for diverse experimental designs .

How can researchers generate and validate cell-specific endoglin knockout mice?

To generate cell-specific endoglin knockout mice, researchers can use the Cre-loxP recombination system as demonstrated in the case of HSC-specific deletion:

• Mouse breeding strategy:

  • Cross mice with a floxed endoglin gene (Eng^f/f) with tissue-specific Cre-expressing mice

  • For HSC-specific deletion, GFAP-Cre mice were used (GFAP^Cre(+)Eng^ΔHSC)

• Validation methods:

  • Genomic PCR validation: Use specific primers to detect wild-type, floxed, and deleted Eng alleles

  • Protein expression analysis: Western blot analysis of isolated target cells

  • Functional assays: Test for expected phenotypic changes in target tissues

• Validation challenges:

  • Complete genetic recombination is rarely achieved (70% protein reduction was observed in HSCs)

  • Expect some residual endoglin expression even in successfully targeted cells

  • Consider using additional markers to confirm cell-specific targeting

• Cell isolation for validation:

  • FACS-sorting of primary cells is recommended for accurate assessment of recombination efficiency

  • Compare protein expression between Cre-negative and Cre-positive animals

What approaches can be used to identify endoglin-positive hematopoietic stem cells in mouse bone marrow?

To identify and isolate endoglin-positive hematopoietic stem cells from mouse bone marrow, researchers can employ a multi-step approach:

• Initial enrichment using the side-population (SP) technique:

  • Utilize Hoechst 33342 dye efflux properties to isolate SP cells

  • SP cells are enriched for hematopoietic stem cell activity

• Further purification using endoglin as a marker:

  • Endoglin-positive cells account for approximately 20% of total SP cells

  • These cells contain all the long-term repopulating (LTR) HSC activity within bone marrow SP

• Transgenic reporter approach:

  • For research purposes, using transgenic reporter genes driven by stem cell enhancers can facilitate visualization and isolation

  • This can be combined with endoglin staining for higher purity

• RNA analysis of rare HSC populations:

  • For gene expression profiling of these rare cells, implement the "constant-ratio PCR" protocol

  • This allows identification of differentially expressed genes using as little as 1 ng of total RNA

  • This approach enables molecular characterization of endoglin-positive HSCs without requiring large cell numbers

How does endoglin interact with the TGF-β and BMP signaling pathways in mouse models?

Endoglin functions as an auxiliary receptor in the TGF-β superfamily signaling complex, with several distinct interaction patterns:

• Receptor associations:

  • Endoglin associates with TGF-β RII, Activin RIIA or RIIB, BMPR-IA/ALK-3, or BMPR-IB/ALK6

  • These associations enhance binding of ligands including Activin A, BMP-2, -7, -9, TGF-β1, and TGF-β3

• BMP9 regulation:

  • BMP9, a circulating cytokine produced in the liver reticuloendothelium and endothelial cells, activates the ALK1-endoglin signaling complex

  • Under BMP9 stimulation, endoglin mRNA and protein levels increase, indicating a positive feedback loop

  • This regulation was observed across multiple primary endothelial cell cultures including human aortic (HAECs), microvascular carotid (HMVEC-C), and umbilical vein endothelial cells (HUVECs)

• TGF-β1 differential responses:

  • Cells with higher endoglin expression show diminished COX-2 expression following TGF-β1 treatment

  • Conversely, human endothelial cells silenced for endoglin expression exhibit higher COX-2 levels after TGF-β1 stimulation

These interactions demonstrate endoglin's complex role in modulating cellular responses to TGF-β family ligands, often acting as a context-dependent regulator of downstream signaling events.

What is the relationship between endoglin and cyclooxygenase-2 (COX-2) in mouse models?

The relationship between endoglin and COX-2 reveals an important regulatory mechanism in vascular biology:

• Inverse correlation in expression levels:

  • Endoglin heterozygous (Eng+/-) mice exhibit specific upregulation of COX-2 in vascular endothelium

  • Transfection of endoglin in L6E9 myoblasts leads to downregulation of COX-2 with no change in COX-1

  • COX-2 promoter activity and protein levels inversely correlate with endoglin levels in doxycyclin-inducible endothelial cells

• Functional consequences:

  • Eng+/- mice show increased urinary excretion of prostaglandin E₂

  • Specific COX-2 inhibition with parecoxib transiently increases arterial pressure in Eng+/- but not in Eng+/+ mice, indicating altered vascular homeostasis

• Interaction with nitric oxide pathway:

  • Chronic NO synthesis inhibition with N^ω-nitro-L-arginine methyl ester (L-NAME) induces marked increase in COX-2 only in normal Eng+/+ mice

  • L-NAME also increases COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells

These findings suggest that endoglin regulates COX-2 expression and activity, and that reduced endoglin levels combined with impaired NO production may contribute to augmented COX-2 expression in Eng+/- mice.

How do findings from endoglin mouse models translate to human disease conditions?

Research on endoglin in mouse models has significant translational relevance to several human conditions:

• Hereditary Hemorrhagic Telangiectasia (HHT):

  • Endoglin heterozygous (Eng+/-) mice serve as a model for HHT type 1, a vascular disorder caused by endoglin haploinsufficiency in humans

  • The vascular abnormalities observed in Eng+/- mice when exposed to VEGF closely resemble those seen in human HHT patients

• Liver fibrosis:

  • Studies in mice with HSC-specific endoglin deletion reveal its protective role against excessive fibrosis

  • Human liver biopsies from patients with acute liver failure and chronic liver injury (NASH, HCV infection) show significantly increased L-Eng expression compared to healthy controls

  • Only patients with acute liver failure showed increased S-Eng levels, suggesting differential regulation of splice variants in different disease contexts

• Stem cell biology:

  • Identification of endoglin as a marker for long-term repopulating hematopoietic stem cells in mice provides a potential marker for human HSC identification and isolation

  • This has implications for bone marrow transplantation and stem cell therapies

These translational connections highlight the value of endoglin mouse models in understanding human disease mechanisms and developing targeted therapies.

What are the current challenges in using endoglin mouse models for therapeutic development?

Researchers working with endoglin mouse models face several key challenges when developing therapeutic applications:

• Incomplete genetic recombination:

  • Even in targeted deletion models, complete endoglin knockout is rarely achieved

  • In HSC-specific deletion models, approximately 70% protein reduction was observed, with residual endoglin expression remaining

  • This partial knockout may complicate interpretation of therapeutic interventions

• Context-dependent signaling effects:

  • Endoglin can either enhance or inhibit signaling through TGF-β receptor complexes depending on cellular context

  • This dual functionality makes it challenging to predict outcomes of endoglin targeting in different tissues or disease states

• Splice variant considerations:

  • The differential expression and regulation of L-Eng and S-Eng splice variants in different disease contexts suggests that therapeutic targeting might need to be splice variant-specific

  • Human and mouse studies show that while L-Eng increases in both acute and chronic liver diseases, S-Eng increases primarily in acute conditions

• Interaction with multiple signaling pathways:

  • Endoglin's involvement in both TGF-β signaling and COX-2 regulation indicates complex pathway interactions

  • Therapeutic modulation might have unintended consequences in related pathways, requiring careful assessment of off-target effects

Understanding these challenges is essential for researchers seeking to translate findings from endoglin mouse models into therapeutic applications for human diseases.