Age-dependent Loss of Hapln1 Erodes Vascular Integrity via Indirect Upregulation of Endothelial ICAM1 in Melanoma

Medical Laboratory Science Geriatrics Medicine Cell Biology Biochemistry Cardiovascular System Oncology Life Sciences Immunology
Hapln1 Melanoma Vascular Integrity ICAM1 Aging Extracellular Matrix

Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma

In a research article titled “Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma” published in Nature Aging in March 2024, significant findings have been revealed. The study, conducted by a team led by Gloria E. Marino-Bravante and Ashani T. Weeraratna from the Department of Biochemistry and Molecular Biology at Johns Hopkins Bloomberg School of Public Health, aims to explore how the age-related loss of HAPLN1 (Hyaluronan and Proteoglycan Link Protein 1) in the melanoma microenvironment affects vascular integrity and its potential therapeutic strategies.

Research Background

Melanoma is the fifth most commonly diagnosed cancer in the United States and the deadliest skin cancer. Statistics show that in the United States alone, an estimated 7,990 individuals die from melanoma each year. As people age, the prognosis of melanoma significantly worsens, with disease recurrence time notably shortening for patients over 55 years old, impacting the five-year survival rate. While the intrinsic impact of aging on cancer progression has long been acknowledged, understanding its complex mechanisms is continuously evolving. Studies have shown that many components of the tumor microenvironment (TME), such as blood vessels, play crucial roles in cancer progression. Therefore, understanding the relationship between aging and melanoma progression is of great significance for developing new therapies.

Research Purpose

Previous studies have suggested that the reduction of HAPLN1 in melanoma-associated vasculature slows cancer progression. HAPLN1 is an essential extracellular matrix protein in the skin’s dermis. Its reduction has been shown to impair melanoma vascular integrity, increasing vascular permeability. The primary aim of this study is to investigate the indirect role of HAPLN1 on endothelial ICAM1 (Intercellular Adhesion Molecule 1) in the melanoma microenvironment of elderly patients and the subsequent impact of ICAM1 on melanoma progression.

Research Methodology

The study delves into the role and mechanisms of HAPLN1 loss in the melanoma microenvironment through a series of experimental techniques. These techniques include immunohistochemistry (IHC), second harmonic generation imaging (SHG), impedance-based assays, and studies using immunocompetent syngeneic mouse models.

Workflow

The research encompassed multiple procedures:

  1. Establishment and Treatment of Mouse Models:

    • Using young syngeneic melanoma (YUMM1.7) cells to establish melanoma models in young (6-8 weeks old) and old (>52 weeks old) mice.
    • Treating older mice with subcutaneous injections of recombinant human HAPLN1 (rHAPLN1) or PBS to observe effects on tumor vascularization.

  2. In Vitro Cell Culture and Matrix Treatment:

    • Conducting 3D vasculogenesis assays in human umbilical vein endothelial cells (HUVECs) embedded in collagen matrices reconstituted by fibroblasts, comparing vasogenic potential under high and low HAPLN1 conditions.

  3. Protein Expression and Vascular Wall Integrity Testing:

    • Assessing VE-cadherin (vascular endothelial cadherin) expression in HUVECs cultured on different matrices to determine vascular wall integrity. Barrier function was evaluated through impedance-based assays and FITC-Dextran leakage experiments.

  4. ICAM1 Expression and Blocking Experiments:

    • Measuring ICAM1 expression to evaluate its role in regulating vascular permeability. Administering ICAM1 blocking antibodies in vivo to observe impacts on melanoma growth and metastasis.

Samples and Techniques

Throughout these processes, sample sizes varied from 3 to 12 per group, employing various experimental techniques such as IHC, immunofluorescence, real-time quantitative PCR (qRT-PCR), and proteomics analysis. Additionally, various gene knockdown strategies (e.g., using shRNA to knock down HAPLN1) were used.

Data Analysis

Data analysis involved calculating the relative intensity of protein expression in images and statistical processing using GraphPad Prism v9 software for various statistical tests, such as t-tests, ANOVA, and non-parametric Mann-Whitney tests.

Research Results

  1. Impact of HAPLN1 on Vasculogenesis and Matrix:

    • In low HAPLN1 environments (old or young mice with HAPLN1 knockdown), HUVECs’ vasogenic potential significantly increased, while in high HAPLN1 environments (young or old + HAPLN1 recombinant treatment), it significantly decreased.
    • Immunohistochemistry showed that fibronectin levels in tumors of older mice were significantly higher than those in younger mice, and recombinant HAPLN1 treatment notably reduced fibronectin levels.

  2. VE-Cadherin Expression and Matrix Stiffness:

    • Endothelial cells cultured on matrices with high HAPLN1 levels exhibited higher VE-cadherin expression, while cells on low HAPLN1 matrices showed lower VE-cadherin levels, highlighting HAPLN1’s crucial role in vascular wall integrity.
    • Impedance-based assays showed that the monolayer resistance value of endothelial cells on high HAPLN1 matrices was higher, indicating improved barrier function.

  3. Role of ICAM1:

    • qRT-PCR and western blot analyses found that ICAM1 expression on low HAPLN1 matrices significantly increased, suggesting that upregulated ICAM1 reduces VE-cadherin expression, affecting vascular permeability.
    • Older mice injected with ICAM1 antibodies showed significantly reduced tumor sizes and metastasis, further confirming ICAM1’s pivotal role in melanoma progression.

Research Significance

The study concludes that the loss of HAPLN1 increases matrix stiffness and indirectly upregulates ICAM1, leading to higher vascular permeability and promoting melanoma metastasis. These findings provide a new perspective on age-related changes in the tumor microenvironment and suggest that targeting ICAM1 could inhibit melanoma progression to some extent, particularly in elderly patients.

Research Highlights

  • Discovered that HAPLN1 indirectly upregulates ICAM1 via a matrix-dependent mechanism, affecting vascular integrity.
  • Proposed new insights into potential therapeutic strategies targeting ICAM1 to improve the prognosis of elderly melanoma patients.
  • Validated the role of HAPLN1 in regulating endothelial cell function in the melanoma microenvironment through multiple in vivo and in vitro experiments.

These findings not only expand the understanding of age-related tumor biology but also provide important clues for developing new targeted therapies. Future studies need to explore the applicability and effectiveness in different tumor types and clinical settings.