Effect of Age-Dependent Loss of HAPLN1 on Vascular Integrity and Melanoma Metastasis

HAPLN1 年龄依赖性缺失对血管完整性和黑色素瘤转移的影响

• 批准号: 10386136
• 负责人: Gloria Elizabeth Marino
• 金额: $ 4.25万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2022-12-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386136
• 关键词: Adherens Junction; Adhesions; Affect; Age; Biological Assay; Biomechanics; Biopsy; Blood Vessels; C57BL/6 Mouse; CD44 Antigens; CD44 gene; Clinical; Collagen; Collagen Receptors; Data; Dermal; Dermis; Disease; Distal; ECM receptor; ENG gene; Endothelial Cells; Endothelium; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fluorescein-5-isothiocyanate; Gel; Hematogenous; Human; Hyaluronan; Hyaluronic Acid; Image; Immunohistochemistry; In Vitro; Incidence; Injections; Intercellular Junctions; Label; Lectin; Lentivirus; Ligands; Ligation; Link; Lymphatic; Lymphatic Endothelial Cells; Lymphatic function; Malignant Neoplasms; Melanoma Cell; Metastatic Melanoma; Microfluidics; Microscopy; Molecular; Mus; Neoplasm Metastasis; Outcome; Patients; Persons; Pharmacology; Phenotype; Prognosis; Prognostic Factor; Proteins; Proteoglycan; Recombinant Proteins; Recombinants; Role; Route; Skin; Skin Aging; Skin Cancer; Structure; Survival Rate; United States; Vascular Permeabilities; adhesion receptor; age effect; age related; aged; angiogenesis; base; cadherin 5; clinical prognostic; density; electric impedance; improved; in vivo; link protein; lymphatic vessel; melanoma; monolayer; neutralizing antibody; older patient; receptor; receptor binding; small hairpin RNA; subcutaneous; tumor; tumor microenvironment

The survival rate for metastatic melanoma is only 23%. Melanoma patients over 55 years old have higher incidence of metastasis than younger patients, independent of other clinical prognostic factors. Our lab has shown that age-dependent changes in the tumor microenvironment are sufficient to drive this disparity. In particular, loss of a secreted extracellular matrix protein called HAPLN1 in the aged dermis confers increased metastasis. HAPLN1 loss coincides with matrix breakdown, which we have shown is sufficient to decrease the barrier function of lymphatic vessels. However, we do not know whether these changes impact the barrier function of blood vessels, and how this affects intravasation and metastasis of melanoma cells via this route. My preliminary studies show that aged mice grow melanoma tumors with significantly more blood vessels compared to young mice. In addition, these vessels have significantly decreased expression of VE-cadherin, which is an endothelial cell junctional protein critical for maintaining vascular integrity. However, treating aged mice with recombinant HAPLN1 is sufficient to rescue these phenotypes. Expression of VE-cadherin is regulated by positive adhesion receptor/extracellular matrix ligand interactions. Accordingly, we have identified two endothelial adhesion receptors which are downregulated in the context of aged ECM and whose expression is dependent on the presence of HAPLN1. Interestingly, we have also shown that aged mice lose the collagen surrounding their blood vessels, which normally functions to support vascular integrity and act as an adhesion ligand. Finally, we have shown that endothelial barrier function in vitro is maintained by presence of HAPLN1 in their substrate matrix. I hypothesize that the ECM structural deficiencies caused by age-dependent loss of HAPLN1 compromise vascular integrity via aberrant adhesion receptor/ECM interactions and increase hematogenous intravasation. To investigate this hypothesis, I will carry out the following aims. Aim 1: Identify the mechanism by which HAPLN1 loss reduces blood vessel integrity. I will determine whether receptor/ligand interactions are required to modulate VE-cadherin expression in a HAPLN1-dependent manner via proximal ligation assay and immunofluorescent microscopy. I will also analyze the effect of HAPLN1 on vascular integrity using an in vitro microfluidics platform. Aim 2: Determine the impact of HAPLN1 on melanoma cell intravasation in vitro and in vivo. I will assess the contribution of HAPLN1 to in vitro intravasation of melanoma cells using an “upside down” intravasation assay. I will assess HAPLN1’s role in in vivo intravasation via vascular imaging. Finally, I will analyze associations between HAPLN1, VE-cadherin, and angiogenesis, and their relationship to age and outcome in human melanoma biopsies. This study will determine the mechanism by which dermal extracellular matrix structure contributes to age-related melanoma progression, which has direct potential to improve the prognoses of older patients.

转移性黑色素瘤的存活率仅为23％。 55岁以上的黑色素瘤患者的患者较高 转移的发生率比年轻患者，与其他临床预后因素无关。我们的实验室有 表明肿瘤微环境的年龄依赖性变化足以驱动这种差异。 特别是，在老年真皮供词中，分泌的细胞外基质蛋白称为hapln1增加 转移。 HAPLN1损失与基质分解相吻合，我们已经证明这足以减少 淋巴视频的屏障功能。但是，我们不知道这些变化是否会影响障碍 血管的功能，以及这如何影响黑色素瘤细胞通过这种途径的转移和转移。我的 初步研究表明，老年小鼠与比较血管明显更多的血管生长黑色素瘤 给年轻的老鼠。此外，这些血管的表达显着降低，这是 内皮细胞连接蛋白对于维持血管完整性至关重要。但是，用 重组Hapln1足以营救这些表型。 VE-钙粘蛋白的表达受 正粘附接收器/细胞外基质配体相互作用。彼此之间，我们已经确定了两个 内皮粘附受体在老化的ECM的背景下被下调，其表达是 取决于Hapln1的存在。有趣的是，我们还表明老年小鼠失去了胶原蛋白 围绕其血管，通常起作用以支持血管完整性并充当粘附 配体。最后，我们已经表明，体外内皮屏障功能是通过在 他们的底物矩阵。我假设ECM结构缺陷是由年龄依赖的损失引起的 通过异常的粘合受体/ECM相互作用，HAPLN1妥协血管完整性并增加 血源性静脉注射。为了调查这一假设，我将执行以下目标。目标1：识别 HAPLN1损失降低血管完整性的机制。我将确定是否 需要接收器/配体相互作用以以Hapln1依赖性方式调节VE-钙粘蛋白的表达 通过近端连接测定和免疫荧光显微镜。我还将分析hapln1对 使用体外微流体平台的血管完整性。目标2：确定Hapln1对 黑色素瘤细胞体外和体内的侵入。我将评估Hapln1对体外的贡献 黑色素瘤细胞使用“倒置”插入测定法的静脉内伸入。我将评估Hapln1在 通过血管成像进行体内插入。最后，我将分析HAPLN1，VE-钙粘蛋白和 血管生成及其与人类黑色素瘤活检年龄和结果的关系。这项研究将确定 皮肤外基质结构有助于年龄相关的黑色素瘤进展的机制， 这具有改善老年患者预后的直接潜力。