MCP 1 Rat

What is MCP-1/CCL2 and what is its function in rats?

MCP-1 (Monocyte Chemotactic Protein-1), also known as CCL2, is a chemokine produced by injured or infected tissues in rats. It serves as a critical signaling molecule that functions through the CCR2 and CCR4 G protein-coupled receptors to recruit specific immune cells—including memory T cells, monocytes, and dendritic cells—to sites of inflammation . With a molecular weight of 14.1 kDa and consisting of 125 amino acids, rat MCP-1 plays fundamental roles in inflammatory responses, tissue repair, and various pathological processes involving immune cell trafficking .

How do you detect and quantify MCP-1 levels in rat biological samples?

MCP-1 levels in rat biological samples can be quantified using enzyme-linked immunosorbent assay (ELISA) techniques. The standard procedure involves generating a calibration curve by plotting the average absorbance obtained for each standard concentration against the corresponding rat MCP-1 concentration (pg/mL). For serum and plasma samples, values interpolated from the standard curve should be multiplied by 25 to calculate the actual pg/mL of rat MCP-1. If further sample dilution was performed, an additional multiplication by the total dilution factor is necessary .

Recovery studies demonstrate that the spike recovery of recombinant rat MCP-1 in various biological matrices (serum and plasma samples) typically ranges from 89-112%, depending on the sample type and spike level, confirming assay reliability :

What are the primary biological sources of MCP-1 in rat models?

In rat models, MCP-1 is primarily produced by multiple cell types in response to tissue injury or infection. Vascular smooth muscle cells (VSMCs) in the arterial walls are significant producers of MCP-1, particularly in aged rats where expression levels are elevated . Additionally, synovial tissue fibroblasts produce substantial amounts of MCP-1 in inflammatory conditions such as rheumatoid arthritis models . The production of MCP-1 is often upregulated in response to pro-inflammatory stimuli, including angiotensin II, which increases with age in rat arterial walls .

How should recombinant rat MCP-1 be reconstituted and stored for experimental use?

For optimal experimental results, lyophilized recombinant rat MCP-1 should be centrifuged in its vial before opening. When reconstituting the product, gently pipet and wash down the vial sides to ensure complete protein recovery into solution. The recommended approach is to reconstitute the lyophilized product with sterile water to a concentration of 0.1 mg/ml, which can be further diluted into other aqueous solutions as needed .

For storage stability, the lyophilized product remains viable for 12 months from the date of receipt when stored at -20°C to -80°C. After reconstitution, the protein remains stable for approximately 1 month when stored at 4°C . Proper adherence to these storage and handling protocols ensures maintenance of MCP-1 biological activity for research applications.

How does MCP-1 expression and signaling change with aging in rat vascular systems?

MCP-1 and its receptor CCR2 show significant upregulation in the aortas of aged rats compared to younger counterparts. Studies using F344×BN rats demonstrate that both MCP-1 and CCR2 mRNAs and proteins increase substantially in old (30-month) versus young (8-month) rat aortas in vivo . Immunohistochemical analysis reveals that cellular MCP-1 and CCR2 staining colocalizes with α-smooth muscle actin in the thickened aortas of older rats . This age-associated increase in the MCP-1/CCR2 axis contributes to vascular remodeling through multiple mechanisms, including enhanced VSMC migration and invasion capabilities.

The mechanistic pathway involves increased angiotensin II levels in aged rat arteries, which induces MCP-1 expression, creating a causal chain from aging to vascular remodeling . VSMCs isolated from older rat aortas express higher levels of MCP-1 and CCR2, and exhibit enhanced migratory and invasive properties compared to VSMCs from younger rats, properties that can be abolished by vCCI, an inhibitor of CCR2 signaling .

What role does MCP-1 play in rat models of rheumatoid arthritis, and how can its inhibition be therapeutically leveraged?

In rat models of adjuvant-induced arthritis (AIA), MCP-1 plays a crucial role in disease progression by promoting leukocyte migration into synovial tissue. The selective inhibition of MCP-1 using a novel inhibitor P8A-MCP-1 in post-onset treatment significantly improves clinical signs of arthritis and histological markers of joint destruction .

Mechanistically, P8A-MCP-1 treatment reduces:

• Joint inflammation and bony erosion

• Monocyte migration into affected tissues

• Proinflammatory cytokine levels (TNF-α, IL-1β)

• Vascular endothelial growth factor levels

• p38 MAPK activation in the joint

This approach demonstrates superior efficacy compared to inhibitors of other chemokines such as RANTES/CCL5. Treatment with the CCR1/CCR5 receptor antagonist methionylated-RANTES or dominant-negative inhibitor for RANTES (44AANA47-RANTES) showed no significant effect on clinical signs of arthritis when administered after disease onset . Even combination therapy with 44AANA47-RANTES plus P8A-MCP-1 did not provide additional benefit beyond P8A-MCP-1 monotherapy, highlighting the specific importance of the MCP-1/CCR2 pathway in this disease model .

How do VSMCs from young rats respond to MCP-1 stimulation compared to VSMCs from older rats?

VSMCs isolated from young rat aortas demonstrate a distinct response pattern to MCP-1 stimulation compared to their aged counterparts. When young VSMCs are treated with MCP-1, they exhibit increased migration and enhanced ability to invade synthetic basement membrane, effectively acquiring characteristics similar to VSMCs from older rat aortas . This MCP-1-dependent VSMC invasiveness can be blocked by vCCI, an inhibitor of CCR2 signaling, confirming the specificity of the effect .

Most notably, after MCP-1 treatment, the migration and invasion capacities of VSMCs from young aortas become indistinguishable from those of VSMCs isolated from older rats . This finding suggests that MCP-1 stimulation effectively induces a phenotypic shift in young VSMCs that mimics age-associated changes, making MCP-1 a potential mediator of vascular aging. The phenomenon provides insight into molecular mechanisms underlying age-related vascular remodeling and identifies potential intervention targets.

What are the optimal experimental conditions for measuring MCP-1-induced chemotaxis in rat cell models?

For accurate assessment of MCP-1-induced chemotaxis in rat cell models, researchers should implement a carefully controlled experimental design. The activity of rat MCP-1 can be determined by its ability to induce chemotaxis of THP-1 cells, with migration quantified using luminescent substrates . Based on established protocols, migration over basal levels is typically observed in response to rat MCP-1 starting at concentrations of 10 ng/ml .

For dilution linearity in experimental design, it's important to note that rat MCP-1 maintains consistent recovery percentages across various dilution ranges:

This dilution linearity data ensures that researchers can appropriately adjust sample concentrations while maintaining proportional measurement accuracy .

How can researchers differentiate between the effects of MCP-1 and other chemokines in rat inflammatory models?

Differentiating between MCP-1 and other chemokines in rat inflammatory models requires selective inhibition strategies combined with comprehensive outcome measurements. Comparative studies using specific inhibitors provide the most direct evidence of differential contributions. For example, in rat AIA models, postonset treatment with P8A-MCP-1 (MCP-1 inhibitor) significantly improved arthritis symptoms, while neither 44AANA47-RANTES (RANTES inhibitor) nor methionylated-RANTES (CCR1/CCR5 receptor antagonist) showed therapeutic efficacy .

Researchers should implement a multi-parameter assessment approach that includes:

• Clinical scoring of disease progression

• Histological evaluation of tissue damage and cellular infiltration

• Immunohistochemistry to identify specific cell populations

• ELISA quantification of inflammatory markers

• Real-time RT-PCR for gene expression analysis

• Western blotting to assess signaling pathway activation

The combination of selective inhibition with comprehensive outcome assessment allows researchers to attribute specific pathological processes to MCP-1 versus other chemokines. This approach revealed that MCP-1 inhibition uniquely reduced p38 MAPK activation and proinflammatory cytokine expression in the rat AIA model, distinguishing its role from that of RANTES .

What are the potential cross-reactivity concerns when studying rat MCP-1 in experimental settings?

When designing experiments to study rat MCP-1, researchers must consider potential cross-reactivity with other chemokines and across species. Specifically formulated rat MCP-1 ELISA kits demonstrate no significant cross-reactivity with related molecules including rat GRO/KC, IL-10, MIP-1α, RANTES, mouse MCP-1, or human MCP-1 . This specificity is crucial for accurate interpretation of results in complex biological samples where multiple chemokines may be present.

For interspecies studies, it's important to note that rat and human MCP-1 share structural similarities but have distinct species-specific regions that affect receptor binding and biological activity. Consequently, researchers should avoid using human MCP-1 inhibitors or antibodies when studying rat models unless cross-reactivity has been explicitly validated. The amino acid sequence of rat MCP-1 (QPDAVNAPLT CCYSFTGKMI PMSRLENYKR ITSSRCPKEA VVFVTKLKRE ICADPNKEWV QKYIRKLDQN QVRSETTVFY KIASTLRTSA PLNVNLTHKS EANASTLFST TTSSTSVEVT SMTEN) should be considered when designing targeting approaches .

How should researchers account for baseline MCP-1 variations in different rat strains and ages?

Baseline MCP-1 levels vary significantly across different rat strains and age groups, necessitating careful experimental design and appropriate controls. When studying age-related effects, researchers should establish strain-specific reference ranges for different age cohorts. For instance, studies comparing young (8-month) versus old (30-month) F344×BN rats have documented substantial differences in baseline aortic MCP-1 expression .

Methodologically, researchers should:

• Include age-matched controls from the same strain when comparing experimental interventions

• Consider potential strain-specific responses when selecting rat models (e.g., F344, Sprague-Dawley, Wistar)

• Establish standardized collection procedures for biological samples, as handling can affect chemokine levels

• Perform preliminary studies to determine baseline variations within experimental colonies

• Include appropriate statistical analyses that account for age and strain as potential covariates

Additionally, when interpreting experimental results from rheumatoid arthritis models, researchers should consider that baseline and induced MCP-1 levels may vary significantly between commonly used strains, potentially affecting therapeutic responses to MCP-1 inhibition .

What are the most reliable methods for studying MCP-1/CCR2 signaling pathways in rat cells?

Studying MCP-1/CCR2 signaling pathways in rat cells requires a combination of molecular, cellular, and pharmacological approaches. Most reliable experimental methods include:

• Receptor antagonist studies: Using vCCI (viral CC chemokine inhibitor) to block CCR2 signaling provides mechanistic insights into MCP-1-dependent processes, as demonstrated in VSMC migration studies .

• Phosphorylation assays: Western blot analysis of p38 MAPK activation (phospho-p38) following MCP-1 stimulation or inhibition reveals downstream signaling events. In arthritic rat models, P8A-MCP-1 treatment decreased p38 MAPK activation in joint tissues .

• Chemotaxis assays: Quantification of THP-1 cell migration using luminescent substrates provides functional assessment of MCP-1 activity, with migration typically detected at concentrations starting from 10 ng/ml .

• Gene expression analysis: Real-time RT-PCR to measure CCR2 receptor expression levels in different tissues or following interventions helps establish correlation between receptor availability and biological responses .

• Co-immunoprecipitation: To identify protein-protein interactions in the CCR2 signaling cascade following MCP-1 binding.

• Cell-specific knockdown or knockout models: Using siRNA or CRISPR-Cas9 approaches to modulate MCP-1 or CCR2 expression in specific rat cell populations.

These complementary approaches provide comprehensive insights into the MCP-1/CCR2 signaling axis in rat models.

How might the MCP-1/CCR2 axis be targeted for age-related vascular diseases in translational rat models?

Based on findings that MCP-1 and CCR2 increase with age in rat aortas and contribute to vascular remodeling, several promising therapeutic approaches are emerging for age-related vascular diseases:

• Selective CCR2 antagonists: Development of small molecule inhibitors with high specificity for rat CCR2 could block age-associated vascular remodeling by preventing MCP-1-induced VSMC migration and proliferation .

• MCP-1 mutant proteins: Building on the success of P8A-MCP-1 in arthritis models, engineered MCP-1 variants could competitively inhibit endogenous MCP-1 binding to CCR2 in vascular tissues .

• Upstream regulation: Since angiotensin II induces MCP-1 expression and increases with age, combining angiotensin-converting enzyme inhibitors or angiotensin receptor blockers with direct MCP-1/CCR2 targeting might provide synergistic benefits .

• Tissue-specific delivery systems: Nanoparticle-based delivery of MCP-1/CCR2 inhibitors specifically to vascular tissues could maximize therapeutic effects while minimizing systemic immunosuppression.

• Epigenetic modulation: Investigating age-related epigenetic changes that regulate MCP-1/CCR2 expression could identify novel targets for intervention in vascular aging.

These approaches require further validation in rat models before potential clinical translation, but they represent promising directions for addressing the significant contribution of MCP-1/CCR2 signaling to age-associated vascular pathology.

What are the methodological considerations for studying MCP-1 in rat models of neuroinflammation and neurodegeneration?

While the provided search results don't specifically address neuroinflammation, the methodological approaches for studying MCP-1 in rat models of neuroinflammation and neurodegeneration can be extrapolated from existing research in other systems. Key considerations include:

• Blood-brain barrier (BBB) permeability: Researchers must account for BBB penetration when administering MCP-1 inhibitors or measuring MCP-1 levels in cerebrospinal fluid versus serum/plasma. Standard dilution protocols for serum samples (1:2, 1:4, 1:8) may need adjustment for central nervous system (CNS) samples .

• Cell-specific effects: The response to MCP-1 varies between microglia, astrocytes, neurons, and infiltrating immune cells. Techniques that distinguish MCP-1 production and CCR2 expression among these populations are essential, similar to the colocalization studies performed in vascular tissue .

• Chronic versus acute models: Neurodegeneration typically involves chronic inflammation, while many experimental models induce acute responses. Long-term studies examining persistent MCP-1/CCR2 signaling would more accurately reflect neurodegenerative conditions.

• Regional variation: Different brain regions may exhibit varied baseline expression and responses to MCP-1. Stereotactic approaches for localized measurement or intervention should be considered.

• Behavioral correlates: Unlike peripheral inflammation models, neuroinflammation studies should include behavioral assessments to correlate MCP-1 levels or interventions with functional outcomes.