ANGPTL7 Mouse

What is the molecular structure and expression pattern of ANGPTL7 in mice?

ANGPTL7 (Angiopoietin-like Protein 7), also known as cornea-derived transcript 6 (CDT6), is a secreted 45 kDa protein that contains a coiled-coil domain (amino acids 30-110) and a fibrinogen-like domain (amino acids 113-334) . The protein is primarily expressed in the stromal layer of the cornea where it forms disulfide-linked homotetramers . Mouse ANGPTL7 shares 88% sequence identity with human ANGPTL7 and 98% with rat ANGPTL7 . Expression has been detected in eye tissue, particularly the cornea, and it plays a role in the bone marrow microenvironment supporting hematopoietic stem cell maintenance .

What are the primary physiological functions of ANGPTL7 in mice?

ANGPTL7 contributes to corneal morphogenesis by suppressing angiogenesis and promoting the deposition of extracellular matrix components . It plays a crucial role in intraocular pressure (IOP) regulation, with knockout mice exhibiting approximately 2 mmHg lower basal IOP compared to wild-type counterparts . Additionally, ANGPTL7 is required for hematopoietic stem cell (HSC) repopulation in a non-cell autonomous manner, as wild-type HSCs fail to properly repopulate in the bone marrow of Angptl7-null mice after serial transplantations .

How do mouse Angptl7 knockout models differ phenotypically from wild-type mice?

Angptl7 knockout mice exhibit several distinct phenotypic differences from wild-type mice:

What are the optimal methods for generating and validating Angptl7 knockout mouse models?

Generation of Angptl7 knockout models has been successfully achieved through TALEN-mediated gene targeting and through embryonic stem cell modifications as in the Angptl7 tm1Lex model (Taconic Biosciences) . Validation requires proper genotyping using PCR with specific primers for the targeted allele. For the tm1Lex model, QIAGEN PCR reagents were used according to the manufacturer's protocol . Expression validation can be performed using quantitative PCR with TaqMan probes (catalog number Mm01256626_m1 for mouse Angptl7) . Western blot analysis using ANGPTL7-specific antibodies should show absence of the 45 kDa band in knockout tissues compared to wild-type controls .

What techniques are recommended for measuring intraocular pressure in Angptl7 mouse models?

The recommended technique for measuring IOP in Angptl7 mouse models involves using a rebound tonometer, such as the TONOVET TV01 (Icare, Vantaa, Finland) . The protocol includes:

• Measurements should be taken between 8 AM and 12 PM to control for diurnal variations

• Induce unconsciousness using isoflurane

• Place mice on a platform with a nose cone and check for lack of blinking response

• Position the tonometer approximately 4 mm away from the central cornea in a horizontal position

• Take at least three measurements per eye (right eye first, then left)

• Calculate the average of measurements to determine IOP for each eye
  This method provides consistent and reliable measurements for comparing wild-type, heterozygous, and knockout animals .

How should researchers design transplantation experiments to study ANGPTL7's role in hematopoietic stem cell regulation?

When designing transplantation experiments to study ANGPTL7's role in HSC regulation, researchers should follow this methodological approach:

• Generate appropriate donor and recipient combinations:

  • Wild-type donors into Angptl7-/-, Angptl7+/-, and wild-type recipients to test microenvironment effects

  • Angptl7-/-, Angptl7+/-, and wild-type donors into wild-type recipients to test cell-intrinsic effects

• Perform sequential transplantations:

  • Primary transplantation to assess initial repopulation

  • Secondary transplantation to test long-term reconstitution capacity

• Measure reconstitution efficiency through peripheral blood chimerism at multiple time points post-transplantation

• Analyze HSC compartments through flow cytometry to determine frequency and absolute numbers of long-term HSCs
  This design allows researchers to distinguish between cell-autonomous and non-cell-autonomous effects of ANGPTL7 on HSC function .

How does ANGPTL7 mechanistically contribute to IOP regulation and glaucoma protection?

ANGPTL7 has emerged as an important regulator of IOP and a potential therapeutic target for glaucoma. Mechanistically, loss-of-function variants in ANGPTL7 are associated with protection from glaucoma and reduced IOP . Studies in knockout mice show that Angptl7 deficiency results in lower (~2 mmHg) basal IOP compared to wild-type mice . Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases IOP .
The mechanism likely involves ANGPTL7's effect on extracellular matrix composition and trabecular meshwork function. ANGPTL7 accumulates in the aqueous humor in glaucoma patients and may promote extracellular matrix deposition that restricts aqueous humor outflow . Both acute silencing of Angptl7 in adult mice and constitutive knockout produce similar IOP-lowering effects (2-4 mmHg), suggesting that ANGPTL7 inhibition could be a viable therapeutic approach for maintaining healthy IOP levels .

What is the relationship between ANGPTL7 and dexamethasone-induced IOP elevation?

Dexamethasone (Dex) treatment is known to elevate IOP, a side effect that can lead to steroid-induced glaucoma. Research indicates that ANGPTL7 may be involved in this process. Studies have examined IOP and outflow facility in Angptl7 knockout mice with and without dexamethasone treatment . Additionally, ANGPTL7 has been quantified in conditioned media from human trabecular meshwork cells in response to Dex, in effluent from perfused human donor eyes, and in aqueous humor from human patients treated with steroids .
The molecular connection likely involves glucocorticoid response elements in the ANGPTL7 promoter region, making it responsive to steroid treatment. Understanding this relationship could lead to interventions that prevent steroid-induced IOP elevation without compromising the therapeutic benefits of corticosteroids.

How does ANGPTL7 function in the bone marrow niche to regulate hematopoietic stem cells?

ANGPTL7 plays a crucial role in the bone marrow niche supporting HSC maintenance and regeneration in a non-cell autonomous manner. Research demonstrates that:

• Wild-type HSCs fail to properly repopulate in the bone marrow of Angptl7-null mice after serial transplantations

• The frequencies and numbers of long-term HSCs in Angptl7-/- recipients are significantly lower than in Angptl7+/- and wild-type recipients

• The engraftment of Angptl7-deficient HSCs in wild-type mice is not impaired, indicating that ANGPTL7 expression in HSCs themselves is not required for their function

• BM cells from primary Angptl7-/- recipients show significantly decreased repopulating activity in secondary transplantation
  These findings suggest that ANGPTL7 in the bone marrow microenvironment is indispensable for supporting HSC repopulation . The exact signaling mechanisms and cellular interactions through which ANGPTL7 affects HSCs remain to be fully elucidated and represent an important area for future research.

What are the optimal conditions for detecting ANGPTL7 in mouse tissues?

Detection of ANGPTL7 in mouse tissues requires specific methodologies for optimal results:
For Western blot analysis:

• Use PVDF membranes probed with Mouse Angiopoietin-like Protein 7/ANGPTL7 Antigen Affinity-purified Polyclonal Antibody (1 μg/mL concentration)

• Follow with HRP-conjugated Anti-Goat IgG Secondary Antibody

• ANGPTL7 appears as a specific band at approximately 45 kDa

• Conduct the experiment under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 8)
  For gene expression analysis:

• Use TaqMan assays with specific primers (Mm01256626_m1 for mouse Angptl7)

• Include appropriate housekeeping genes (e.g., Mouse Gapdh, Mm99999915_g1) for normalization
  Proper storage of antibodies and proteins is critical:

• Store in manual defrost freezers and avoid repeated freeze-thaw cycles

• Maintain at -20 to -70°C for up to 12 months in original condition

• After reconstitution, store at 2 to 8°C for up to 1 month or at -20 to -70°C for up to 6 months under sterile conditions

How can researchers address variability in IOP measurements when studying Angptl7 mouse models?

Variability in IOP measurements presents a significant challenge in Angptl7 research. To address this issue, researchers should:

• Control for time-of-day effects by conducting all measurements within a consistent time window (e.g., 8 AM to 12 PM)

• Use standardized anesthesia protocols, as anesthetic agents can affect IOP

• Take multiple measurements per eye (minimum of three) and use the average value

• Ensure proper tonometer positioning approximately 4 mm from the central cornea with the probe in a horizontal position

• Employ masked study designs where the experimenter is unaware of the genotype during measurements

• Include adequate sample sizes based on power calculations that account for known variability in mouse IOP measurements

• Consider using longitudinal measurements on the same animals to reduce inter-individual variability
  These methodological considerations help ensure reliable IOP data when comparing wild-type, heterozygous, and knockout Angptl7 mouse models .

What controls should be included when analyzing the effects of ANGPTL7 manipulation on HSC populations?

When analyzing effects of ANGPTL7 manipulation on HSC populations, researchers should include these essential controls:

• Genotype controls:

  • Wild-type littermates (+/+)

  • Heterozygous mice (+/-)

  • Homozygous knockout mice (-/-)

• Transplantation controls:

  • Reciprocal transplantation designs (all combinations of donor/recipient genotypes)

  • Multiple cell doses to assess dose-dependent effects

  • Competitive repopulation assays with defined ratios of test and competitor cells

• Analysis controls:

  • Multiple timepoints post-transplantation (4, 8, 12, and 16 weeks)

  • Assessment of multi-lineage reconstitution (myeloid, B-cell, T-cell)

  • Secondary transplantation to test long-term self-renewal capability

• Technical controls:

  • Consistent flow cytometry gating strategies

  • Standardized cell isolation procedures

  • Proper irradiation controls to ensure complete myeloablation
    This comprehensive control strategy enables robust analysis of ANGPTL7's effects on HSC function while minimizing experimental artifacts and misinterpretation .

How might ANGPTL7-targeting therapies be developed for glaucoma treatment?

Development of ANGPTL7-targeting therapies for glaucoma represents a promising research direction. Based on current evidence, several approaches warrant investigation:

• Anti-ANGPTL7 neutralizing antibodies have shown efficacy in lowering IOP in experimental models:

  • Intravitreal injection of neutralizing antibodies reduced IOP in rabbits

  • Antibodies tested in 3D culture of outflow cells and perfused human donor eyes demonstrated efficacy

• RNA interference strategies:

  • Acute Angptl7 silencing in adult mice lowered IOP by 2-4 mmHg

  • siRNA or antisense oligonucleotides could provide temporally controlled ANGPTL7 inhibition

• Small molecule inhibitors:

  • Targeting ANGPTL7's functional domains or its interaction with extracellular matrix components

  • High-throughput screening of compound libraries against ANGPTL7 activity

• Gene therapy approaches:

  • CRISPR-based gene editing to introduce protective loss-of-function variants

  • AAV-delivered ANGPTL7 shRNA for sustained local inhibition
    The development pathway should include in vitro efficacy testing, preclinical animal studies with careful IOP monitoring, and ultimately human trials focusing on both efficacy in IOP reduction and safety profiles, particularly regarding corneal health and integrity .

What are the potential applications of ANGPTL7 in hematopoietic stem cell transplantation and regenerative medicine?

ANGPTL7's role in HSC regulation suggests several potential applications in transplantation and regenerative medicine:

How might cross-talk between ANGPTL7's functions in different tissues impact therapeutic targeting?

ANGPTL7 functions in multiple tissues, including the eye and bone marrow, raising important considerations for therapeutic targeting: