CTGF Human (183-255)

What is CTGF Human (183-255) and which functional domain does it represent?

CTGF Human (183-255) refers to the amino acid sequence spanning positions 183-255 in the human Connective Tissue Growth Factor protein. This sequence corresponds to part of the Thrombospondin Type 1 (TSP1) domain of CTGF, which is located approximately between amino acids Asn198-Glu243 according to established domain boundaries . The TSP1 domain is one of four modular domains that comprise the complete CTGF protein structure, alongside the insulin-like growth factor-binding protein (IGFBP) domain, von Willebrand factor type C repeat (VWC) domain, and cysteine knot-containing carboxyl domain (CT) .

The TSP1 domain contains the characteristic "CSXXCG" sequence and six conserved cysteine residues that are critical for its three-dimensional structure. Specific amino acids within this region—namely Trp206, Ser218, Arg220, Gln233, and Arg235—are 100% conserved across all CCN family proteins, highlighting their functional importance .

What are the key structural features of the TSP1 domain in CTGF?

The TSP1 domain of CTGF exhibits several distinctive structural characteristics:

• Conserved motifs: Contains the "CSXXCG" amino acid sequence motif, which is characteristic of thrombospondin type 1 repeats

• Cysteine framework: Features six conserved cysteine residues that form intramolecular disulfide bonds critical for proper protein folding and stability

• Highly conserved residues: Five amino acids (Trp206, Ser218, Arg220, Gln233, and Arg235) are perfectly conserved across all CCN family proteins, suggesting their essential role in domain function

• Binding interfaces: Contains specific regions that mediate interactions with VEGF, particularly binding to the exon 7-coded region of VEGF165

• Integrin interaction sites: Possesses structural elements that facilitate binding to cell surface integrins, particularly α6β1

These structural features collectively enable the TSP1 domain to mediate protein-protein interactions crucial for CTGF's diverse biological functions.

What biological functions are associated with the TSP1 domain of CTGF?

The TSP1 domain mediates several key biological functions of CTGF:

• VEGF regulation: The TSP1 domain binds directly to the exon 7-coded region of VEGF165, enabling CTGF to negatively regulate angiogenic activity of VEGF. This interaction creates a CTGF-VEGF complex that can be disrupted under specific conditions such as RHOA/Rho-kinase inactivation in mesenchymal stem cells, allowing controlled release of VEGF .

• Cell adhesion: Through interactions with α6β1 integrin and LRP (Low-density lipoprotein Receptor-related Protein), the TSP1 domain mediates cellular adhesion processes which are essential for tissue organization and cell migration .

• Collagen deposition: The TSP1 domain influences extracellular matrix composition by promoting collagen deposition, a process integral to both normal tissue maintenance and pathological fibrosis .

• Signal transduction: By interacting with cell surface receptors, this domain contributes to intracellular signaling pathways that regulate cell behavior, including proliferation and differentiation .

These functions position the TSP1 domain as a critical mediator of CTGF's effects on tissue remodeling, wound healing, and disease processes.

Which cell types primarily express CTGF?

CTGF expression has been documented across diverse cell types:

• Vascular cells: Endothelial cells (where CTGF was first discovered) and vascular smooth muscle cells (VSMCs) express CTGF, with expression patterns changing throughout the progression of fibrotic disease .

• Fibroblasts: These cells are major producers of CTGF, particularly when activated by TGF-β or mechanical stress. Fibroblasts cultured on stiff substrates show significantly higher CTGF expression than those on physiologically soft matrices .

• Stellate cells: These specialized cells, particularly in the liver, are known to produce CTGF .

• Tumor cells: Various cancer cells express CTGF, which can influence tumor progression .

• Muscle cells: Both cardiac and skeletal muscle cells produce CTGF, with implications for cardiac fibrosis and muscular dystrophies .

• Neural cells: CTGF is widely distributed throughout the central nervous system, suggesting roles in neural development and function .

• Ocular tissues: Expression has been detected in cornea, iris, ciliary body, and choroid of the eye .

The cell type-specific expression patterns of CTGF contribute to its varied roles in different tissues and disease contexts.

How is CTGF expression regulated at the transcriptional level?

CTGF expression is regulated through multiple signaling pathways and transcription factors:

• Hippo pathway: The transcription co-activators YAP and TAZ function as downstream effectors of the Hippo pathway. When dephosphorylated, YAP/TAZ translocate to the nucleus and act as transcriptional activators of CTGF. Environmental factors such as temperature and static magnetic fields can influence this pathway, upregulating CTGF by increasing nuclear localization of YAP/TAZ .

• ETS1 transcription factor: ETS1 binds to GGAA sequences in the CTGF proximal promoter to activate transcription. Similar to YAP/TAZ, ETS1 participates in processes including fibrosis, angiogenesis, and osteogenesis .

• SMAD signaling: TGF-β activates SMAD 2/3 through phosphorylation, which then mediates CTGF expression. This represents a major pathway by which TGF-β induces fibrotic responses .

• MAPK pathway: Mitogen-activated protein kinases contribute to CTGF regulation through their activity on various transcription factors .

• Matrix stiffness: Mechanical cues from the extracellular environment significantly impact CTGF expression. Human lung fibroblasts cultured on stiff substrates (50 kPa or tissue culture plastic) show markedly higher CTGF mRNA levels compared to those on soft matrices (1 kPa) that mimic normal lung tissue .

• TGF-β concentration: CTGF mRNA expression increases in a dose-dependent manner with TGF-β stimulation in human lung fibroblasts .

This complex regulatory network allows for context-specific CTGF expression in different physiological and pathological settings.

How does the TSP1 domain of CTGF interact with VEGF and what are the implications for angiogenesis research?

The TSP1 domain of CTGF interacts specifically with VEGF through a direct binding mechanism:

• Binding specificity: The TSP1 domain binds to the exon 7-coded region of VEGF165, forming a CTGF-VEGF complex that modulates VEGF's angiogenic activity .

• Regulatory mechanism: This interaction allows CTGF to function as a VEGF reservoir. Under specific conditions, such as RHOA/Rho-kinase inactivation in mesenchymal stem cells, VEGF can be released from this complex, creating a controlled release system for angiogenic signaling .

• Tissue remodeling: During tissue remodeling processes, the dynamic formation and dissolution of CTGF-VEGF complexes help regulate the spatial and temporal aspects of blood vessel formation .

For angiogenesis research, these interactions have several implications:

• Therapeutic targeting: Understanding this interaction provides opportunities for developing peptides or small molecules that could either disrupt or enhance CTGF-VEGF binding, depending on the desired therapeutic outcome.

• Biomarker potential: The ratio of bound to free VEGF could serve as a marker for disease progression in conditions where abnormal angiogenesis plays a role.

• Drug delivery strategies: The natural release mechanism of VEGF from CTGF complexes could inspire biomimetic drug delivery systems for controlled release of angiogenic or anti-angiogenic agents.

• Cancer research: Since tumor angiogenesis is critical for cancer progression, the CTGF-VEGF interaction may represent an important regulatory node in cancer biology.

Researchers studying angiogenesis should consider incorporating analyses of CTGF-TSP1 domain interactions to gain a more complete understanding of vascular regulation in their experimental systems.

What roles does the TSP1 domain play in fibrotic diseases compared to other CTGF domains?

The TSP1 domain contributes to fibrotic disease progression through mechanisms that complement and differ from other CTGF domains:

• ECM accumulation: The TSP1 domain influences collagen deposition through interactions with α6β1 integrins and LRP, directly contributing to excessive matrix accumulation characteristic of fibrosis .

• Cell adhesion modulation: By mediating adhesion processes, the TSP1 domain affects fibroblast behavior, including migration and transformation to myofibroblasts - key cellular events in fibrotic disease progression .

• VEGF sequestration: The TSP1 domain's ability to bind and sequester VEGF impacts vascular remodeling during fibrosis development, affecting tissue perfusion and inflammatory cell recruitment .

In comparison with other domains:

• IGFBP domain: This domain interacts with insulin-like growth factors and contributes to matrix accumulation in conditions like tubulointerstitial fibrosis, working in parallel with TSP1 domain functions .

• VWC domain: While the TSP1 domain primarily affects VEGF signaling, the VWC domain modulates TGF-β and BMP signaling. The VWC domain enhances TGF-β signaling (a major pro-fibrotic pathway) while typically antagonizing BMP-2 and BMP-7 (which often have anti-fibrotic effects) .

• CT domain: This domain mediates interactions with cell surface integrins and heparan sulfate proteoglycans, affecting cell adhesion and migration in fibrotic processes through mechanisms distinct from the TSP1 domain .

In fibrotic disease models, including TGF-β1-induced lung fibrosis, the coordinated action of all domains appears necessary for full fibrotic response, as evidenced by the partial attenuation of fibrosis achieved with pamrevlumab, a CTGF inhibitory antibody .

How does matrix stiffness influence CTGF expression and particularly the functions mediated by the TSP1 domain?

Matrix stiffness profoundly influences CTGF expression through mechanotransduction mechanisms:

Implications for TSP1 domain functions:

• Enhanced TSP1-mediated adhesion: Increased CTGF expression on stiff matrices likely enhances TSP1 domain-mediated adhesion to extracellular matrix components, potentially creating a positive feedback loop in fibrotic tissues.

• Altered VEGF binding capacity: The higher concentration of CTGF (and consequently its TSP1 domain) in stiff microenvironments may increase sequestration of VEGF, affecting angiogenic responses in fibrotic tissues.

• Mechanosensing amplification: The TSP1 domain's interactions with integrins may participate in mechanosensing mechanisms, potentially amplifying cellular responses to mechanical signals in progressively stiffening tissues.

These findings suggest that targeting the mechanical aspects of the cellular microenvironment could indirectly modulate TSP1 domain functions by altering CTGF expression levels, presenting a potential therapeutic approach for fibrotic conditions.

What experimental models best represent CTGF-mediated fibrosis for studying the TSP1 domain?

Based on the provided information, several experimental models effectively capture CTGF-mediated fibrotic processes:

• AdTGF-β1-induced rat lung fibrosis model: This model uses an adenoviral vector encoding active TGF-β1 (with mutations at positions 223 and 225) to induce lung fibrosis. Key advantages include:

  • Produces more intense, rapid, and severe fibrotic reactions than traditional bleomycin models

  • Shows significant upregulation of CTGF expression

  • Develops fibrosis beginning at day 7 and persisting until day 28

  • Exhibits less pronounced acute lung injury and inflammation than bleomycin models, allowing better focus on fibrogenic mechanisms

• Cell type-specific in vitro models:

  • Vascular smooth muscle cells (VSMCs): VSMCs isolated from pulmonary arteries in AdTGF-β1 rat lungs show upregulated CTGF expression, particularly at days 7 and 14 after infection, representing early-stage fibrosis

  • Endothelial cells (ECs): Sorted ECs from AdTGF-β1 rats show increased CTGF expression at days 14 and 28, representing middle to late stages of fibrosis

  • Lung fibroblasts on variable stiffness matrices: Culturing human lung fibroblasts on substrates of different stiffness (1 kPa for physiological, 50 kPa for pathological, and tissue culture plastic for extreme stiffness) allows studying how mechanical properties influence CTGF expression and function

• Duchenne muscular dystrophy (DMD) models:

  • The mdx mouse model of DMD shows CTGF-dependent skeletal muscle fibrosis

  • Genetic deletion of CTGF in this model improves muscle strength, reduces fibrosis, and decreases apoptotic damage

For studying TSP1 domain-specific functions:

• These models can be combined with domain-specific blocking approaches (antibodies or peptides targeting the TSP1 domain)

• Experiments comparing wild-type CTGF with TSP1 domain mutants in these systems would help elucidate domain-specific contributions

• Co-immunoprecipitation studies in these models could identify TSP1 domain binding partners in different fibrotic contexts

The time-course studies possible in the AdTGF-β1 model are particularly valuable for understanding how TSP1 domain functions might change throughout fibrosis progression.

What are the current therapeutic approaches targeting CTGF and how might TSP1 domain-specific interventions differ?

Current therapeutic approaches targeting CTGF include:

• Monoclonal antibodies:

  • FG-3019 (pamrevlumab): This antibody has shown promising outcomes in clinical and preclinical trials for various fibrotic conditions. In a phase 2 trial for Duchenne muscular dystrophy, it demonstrated positive effects on pulmonary and cardiac function, as well as preservation of upper limb function .

  • FG-3149: Another anti-CTGF monoclonal antibody in development .

• Angiotensin system modulators:

  • Angiotensin-converting enzyme inhibitors: Drugs like enalapril reduce CTGF levels and improve skeletal muscle fibrosis in mdx mice .

  • AT-1R blockers: These agents not only decrease CTGF levels but also block CTGF activity, suggesting AT-1R is necessary for CTGF's biological response .

• Genetic approaches:

  • Deletion of CTGF expression has been shown to improve muscle strength, skeletal muscle impairment, apoptotic damage, and fibrosis in mdx mice .

Potential TSP1 domain-specific therapeutic approaches could include:

• Domain-specific antibodies: Antibodies specifically targeting the TSP1 domain could block its interactions with VEGF and integrins without affecting other CTGF functions, potentially providing more targeted anti-fibrotic effects with fewer side effects.

• Peptide inhibitors: Synthetic peptides mimicking key regions of the TSP1 domain could compete for binding with natural ligands, selectively inhibiting domain-specific functions.

• Small molecule disruptors: Small molecules designed to bind the TSP1 domain at critical interaction sites could prevent protein-protein interactions essential for its pro-fibrotic effects.

• TSP1-VEGF interaction modulators: Compounds specifically targeting the interface between TSP1 and VEGF could modulate angiogenesis in fibrotic tissues without broadly affecting CTGF functions.

The advantages of TSP1 domain-specific approaches would include:

• Preservation of beneficial functions mediated by other CTGF domains

• Potential reduction in side effects compared to pan-CTGF inhibition

• More precise targeting of specific pathological processes like excessive collagen deposition or abnormal angiogenesis

Current evidence suggests that while whole-CTGF targeting shows promise, domain-specific approaches could offer more tailored therapeutic options for different fibrotic conditions.

What are the optimal methods for isolating and characterizing the TSP1 domain (183-255) of CTGF?

For researchers interested in studying the CTGF TSP1 domain, several methodological approaches are recommended:

• Recombinant protein expression:

  • Bacterial expression systems: Using E. coli with appropriate expression vectors (pET, pGEX) to produce the TSP1 domain with affinity tags (His, GST) for purification

  • Eukaryotic expression systems: HEK293 or CHO cells for expression of properly folded domain with intact disulfide bonds and post-translational modifications

  • Yeast expression systems: Pichia pastoris offers advantages for disulfide-rich proteins like the TSP1 domain

• Purification strategies:

  • Affinity chromatography: Using immobilized metal affinity chromatography (IMAC) for His-tagged proteins or glutathione-sepharose for GST-fusion proteins

  • Size exclusion chromatography: As a polishing step to obtain highly pure protein and confirm monomeric state

  • Ion exchange chromatography: To separate different charge variants that might arise from different folding patterns

• Structural characterization:

  • Circular dichroism (CD) spectroscopy: To confirm proper secondary structure formation

  • Mass spectrometry: For accurate molecular weight determination and identification of post-translational modifications

  • Differential scanning fluorimetry: To assess thermal stability and proper folding

• Functional validation:

  • VEGF binding assays: Using surface plasmon resonance (SPR) or microscale thermophoresis to measure binding kinetics with VEGF165

  • Cell adhesion assays: Quantifying attachment of cells to surfaces coated with the purified TSP1 domain

  • Integrin interaction studies: Pull-down assays with α6β1 integrin to confirm binding capacity

• Domain-specific antibody generation:

  • Development of monoclonal antibodies specifically recognizing the TSP1 domain for immunoprecipitation, Western blotting, and immunofluorescence applications

  • Epitope mapping to ensure antibodies target functional regions within the domain

These methodological approaches provide a comprehensive toolkit for isolating and characterizing the TSP1 domain, enabling detailed investigation of its structural features and functional properties in various experimental contexts.

How can researchers effectively measure CTGF-TSP1 domain interactions with target proteins?

Researchers can employ several methodologies to characterize interactions between the CTGF-TSP1 domain and its binding partners:

• Biophysical interaction analysis:

  • Surface Plasmon Resonance (SPR): Provides real-time, label-free analysis of binding kinetics (association and dissociation rates) and affinities. Immobilize either the TSP1 domain or its binding partner (e.g., VEGF) on a sensor chip and flow the other protein as analyte.

  • Isothermal Titration Calorimetry (ITC): Measures the heat released or absorbed during binding to determine thermodynamic parameters (ΔH, ΔS, ΔG) and stoichiometry.

  • Microscale Thermophoresis (MST): Detects changes in the movement of molecules along microscopic temperature gradients to measure binding affinities in solution.

• Biochemical interaction assays:

  • Co-immunoprecipitation: Using domain-specific antibodies to pull down protein complexes from cell lysates, followed by Western blotting to detect binding partners.

  • Pull-down assays: Immobilizing recombinant TSP1 domain on beads to capture binding partners from cell lysates.

  • ELISA-based binding assays: Direct or competitive ELISAs to quantify protein-protein interactions and screen for inhibitors.

• Cellular interaction visualization:

  • Proximity Ligation Assay (PLA): Detects protein interactions in situ with high specificity and sensitivity, providing spatial information about interactions within cells.

  • Förster Resonance Energy Transfer (FRET): Measures energy transfer between fluorescently labeled proteins when they interact closely.

  • Bimolecular Fluorescence Complementation (BiFC): Split fluorescent protein fragments fused to potential interacting proteins reassemble and fluoresce when brought together.

• Computational approaches:

  • Molecular docking: Predicts potential binding interfaces between the TSP1 domain and partners like VEGF.

  • Molecular dynamics simulations: Models the dynamic behavior of protein complexes over time to identify stable interaction points.

• Peptide mapping:

  • Peptide arrays: Synthesizing overlapping peptides spanning the TSP1 domain to identify specific binding regions.

  • Alanine scanning mutagenesis: Systematically replacing key residues with alanine to identify those critical for binding.

For specific CTGF-TSP1 domain interactions with VEGF, researchers should consider:

• Comparing binding of full-length CTGF versus isolated TSP1 domain

• Examining how matrix stiffness and mechanical forces affect these interactions

• Investigating how disease-related conditions (e.g., high glucose, inflammatory cytokines) modify binding properties

These methodologies provide complementary information to fully characterize the interactions between the CTGF-TSP1 domain and its binding partners in both physiological and pathological contexts.

What cell culture systems best model the effects of CTGF-TSP1 domain in fibrotic disease progression?

Optimal cell culture systems for modeling CTGF-TSP1 domain functions in fibrosis include:

• Mechanically tunable culture systems:

  • Hydrogels with adjustable stiffness: Polyacrylamide or hyaluronic acid-based hydrogels can be tuned to mimic the physiological stiffness of normal tissue (0.2-2 kPa) or the pathological stiffness of fibrotic tissue (2-35 kPa)

  • Micropatterns with controlled cell geometry: Systems that control cell spreading and cytoskeletal tension to modulate mechanotransduction pathways that regulate CTGF expression

• Cell type-specific models:

  • Primary fibroblasts: Isolated from fibrotic human or animal tissues (e.g., IPF lungs or AdTGF-β1-treated rats) maintain disease-specific phenotypes and show elevated CTGF expression

  • Vascular smooth muscle cells (VSMCs): As demonstrated in the AdTGF-β1 model, VSMCs from early fibrotic stages (days 7-14) show increased CTGF expression and can model vascular contributions to fibrosis

  • Endothelial cells: ECs from middle-to-late fibrotic stages (days 14-28) exhibit increased CTGF expression and can model endothelial dysfunction in fibrosis

• Co-culture systems:

  • Fibroblast-epithelial co-cultures: Model epithelial-mesenchymal interactions critical in organs like lung and kidney

  • Fibroblast-endothelial co-cultures: Particularly relevant for studying TSP1-VEGF interactions in the context of fibrosis and angiogenesis

  • Three-dimensional organoids: Incorporating multiple cell types to recapitulate tissue architecture and cell-cell communication

• Dynamic culture conditions:

  • Cyclic stretch systems: Apply mechanical strain to mimic respiratory movements in lung fibrosis or cardiac contractions in heart fibrosis

  • Flow systems: Expose endothelial cells to controlled shear stress to model vascular aspects of fibrotic diseases

  • Hypoxia chambers: Create low oxygen environments that often accompany fibrotic tissue remodeling

• TGF-β stimulation protocols:

  • Dose-response studies: Treating cells with different concentrations of TGF-β (as shown to dose-dependently increase CTGF expression)

  • Pulse vs. chronic exposure: Comparing short-term vs. long-term TGF-β exposure to model acute vs. chronic fibrotic stimuli

• Domain-specific intervention approaches:

  • TSP1 domain overexpression: Transfecting cells with constructs expressing only the TSP1 domain

  • Domain-blocking antibodies: Treating cultures with antibodies specifically targeting the TSP1 domain

  • Competition assays: Using recombinant TSP1 domain or peptide fragments to compete with endogenous CTGF

The AdTGF-β1-induced rat model provides valuable primary cells from different stages of fibrosis progression, enabling researchers to study temporal changes in TSP1 domain function throughout the disease course .

What are the key considerations when designing inhibitors targeting the CTGF-TSP1 domain?

When designing inhibitors specifically targeting the CTGF-TSP1 domain, researchers should consider:

• Target site selection:

  • VEGF binding interface: Designing inhibitors that disrupt the interaction between the TSP1 domain and the exon 7-coded region of VEGF165

  • Integrin binding regions: Targeting sites that mediate interactions with α6β1 integrin to modulate cell adhesion and signaling

  • LRP binding sites: Focusing on regions that interact with LRP to affect collagen deposition

  • Conserved residues: Particular attention to the highly conserved amino acids (Trp206, Ser218, Arg220, Gln233, and Arg235) that may be critical for function

• Inhibitor modality selection:

  • Monoclonal antibodies: Building on the success of pan-CTGF antibodies like FG-3019 (pamrevlumab), but with epitopes specifically within the TSP1 domain

  • Peptide mimetics: Designing peptides that mimic TSP1 binding interfaces to competitively inhibit natural interactions

  • Small molecules: Targeting binding pockets or protein-protein interfaces within the TSP1 domain

  • Aptamers: Developing nucleic acid aptamers with high affinity and specificity for the TSP1 domain

• Selectivity considerations:

  • Cross-reactivity with other CCN family members: Ensuring specificity for CTGF-TSP1 versus similar domains in other CCN proteins

  • Domain selectivity: Avoiding interference with other CTGF domains to maintain beneficial functions

  • Interaction-specific targeting: Designing inhibitors that block specific protein interactions while permitting others

• Delivery strategies:

  • Tissue-specific targeting: Developing delivery systems that preferentially accumulate in fibrotic tissues

  • Cell-specific delivery: Targeting cell types that are major contributors to CTGF expression in specific diseases (e.g., fibroblasts in skin fibrosis, VSMCs in early lung fibrosis, or endothelial cells in later stages)

  • Extracellular vs. intracellular inhibition: Deciding whether to target secreted CTGF or intracellular processes

• Efficacy evaluation:

  • In vitro testing hierarchy: Progressing from biochemical binding assays to cell-based functional assays using mechanical stress models that upregulate CTGF naturally

  • Ex vivo tissue models: Using precision-cut tissue slices from fibrotic organs to test inhibitor effects

  • Animal model selection: Choosing appropriate disease models like the AdTGF-β1-induced lung fibrosis model

• Combination therapy potential:

  • Synergy with TGF-β inhibitors: Assessing combination with upstream inhibitors of the TGF-β pathway

  • Complementary targeting of other domains: Evaluating partial inhibition of multiple domains versus complete inhibition of the TSP1 domain alone

The partial attenuation of fibrosis achieved with pan-CTGF antibodies suggests that domain-specific inhibition might need to be combined with other approaches for maximal therapeutic effect .

How can researchers distinguish between effects mediated by different domains of CTGF in experimental settings?

Distinguishing domain-specific effects of CTGF requires sophisticated experimental approaches:

• Domain deletion mutants:

  • Generate recombinant CTGF proteins with specific domains deleted (ΔTSP1, ΔIGFBP, ΔVWC, ΔCT)

  • Compare functional outcomes of wild-type versus domain-deleted proteins in relevant assays

  • Use in both cell culture and in vivo models to identify domain-specific contributions to biological responses

• Domain swapping experiments:

  • Create chimeric proteins by swapping the TSP1 domain with corresponding domains from other CCN family members

  • Assess whether functional properties track with the origin of the TSP1 domain

  • Identify conserved versus unique functions across family members

• Domain-blocking antibodies:

  • Develop and validate antibodies with epitopes specific to individual domains

  • Apply these antibodies to block domain-specific interactions without affecting other domains

  • Compare effects with pan-CTGF antibodies like FG-3019 to determine domain-specific contributions

• Domain-specific binding partners:

  • Use the known binding partners of each domain as competitive inhibitors

  • For the TSP1 domain, VEGF165 fragments or peptides derived from its exon 7-coded region could selectively compete with TSP1-mediated interactions

  • For other domains, appropriate ligands include IGFs (IGFBP domain), BMPs or TGF-β (VWC domain), and specific integrins (CT domain)

• Site-directed mutagenesis:

  • Create point mutations in key residues specific to each domain

  • For the TSP1 domain, target the highly conserved Trp206, Ser218, Arg220, Gln233, and Arg235

  • Assess how these mutations affect specific functions while leaving other domains intact

• Temporal analysis:

  • Exploit the temporal differences in domain-specific functions

  • In the AdTGF-β1 model, differentiate between early effects (days 7-14) when VSMCs show increased CTGF expression versus later effects (days 14-28) when endothelial cells become major contributors

• Cell type-specific approaches:

  • Utilize cell types that primarily respond to specific domains

  • For TSP1 domain, focus on endothelial cells (VEGF interactions) and cells expressing α6β1 integrin

  • Compare with cells primarily responsive to other domains

• Readout selection:

  • Choose assays that selectively measure functions associated with each domain:

    • TSP1 domain: VEGF sequestration, cell adhesion via α6β1 integrin, collagen deposition

    • IGFBP domain: IGF binding and matrix accumulation

    • VWC domain: TGF-β enhancement and BMP antagonism

    • CT domain: Integrin-mediated adhesion and heparan sulfate proteoglycan binding

• Mechanical environment manipulation:

  • Vary matrix stiffness to differentially regulate CTGF expression

  • Determine if mechanical regulation preferentially affects certain domain-specific functions

These approaches, used in combination, can effectively distinguish the unique contributions of the TSP1 domain from other CTGF domains in experimental systems.