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.

转移性黑色素瘤的生存率仅为23%。55岁以上的黑色素瘤患者 转移发生率高于年轻患者，独立于其他临床预后因素。我们的实验室 研究表明，肿瘤微环境中与年龄相关的变化足以导致这种差异。在 特别地，在老化真皮中被称为HAPLN 1的分泌的细胞外基质蛋白的损失赋予增加的 转移HAPLN 1损失与基质分解相一致，我们已经证明这足以减少 淋巴管的屏障功能。然而，我们不知道这些变化是否会影响屏障 血管的功能，以及这如何通过这一途径影响黑色素瘤细胞的浸润和转移。我 初步研究表明，老年小鼠生长的黑色素瘤肿瘤血管明显多于老年小鼠， 年轻的老鼠。此外，这些血管的VE-钙粘蛋白表达显著降低，这是一个重要因素。 内皮细胞连接蛋白，对维持血管完整性至关重要。然而，用 重组HAPLN 1足以拯救这些表型。VE-钙粘蛋白的表达受 阳性粘附受体/细胞外基质配体相互作用。因此，我们确定了两个 内皮粘附受体，其在老化ECM的情况下下调，并且其表达是 依赖于HAPLN 1的存在。有趣的是，我们还发现老年小鼠失去了胶原蛋白， 它们的血管周围，通常起到支持血管完整性和作为粘附的作用， 配体。最后，我们已经表明，在体外，内皮屏障功能是通过HAPLN 1的存在来维持的。 基质基质。我推测，ECM结构缺陷是由年龄依赖性的细胞外基质丢失引起的。 HAPLN 1通过异常粘附受体/ECM相互作用损害血管完整性，并增加 血行内渗为了研究这个假设，我将实现以下目标。目标1：确定 HAPLN 1缺失降低血管完整性的机制。我将决定 受体/配体相互作用是以HAPLN 1依赖性方式调节VE-钙粘蛋白表达所必需的 通过近端连接测定和免疫荧光显微镜。我还将分析HAPLN 1对 使用体外微流体平台的血管完整性。目标2：确定HAPLN 1对 黑色素瘤细胞内渗的体外和体内研究。我将评估HAPLN 1在体外对 使用“倒置”内渗测定法测定黑色素瘤细胞的内渗。我将评估HAPLN 1在以下方面的作用： 通过血管成像的体内血管内渗。最后，我将分析HAPLN 1、VE-钙粘蛋白和 血管生成，以及它们与年龄和人类黑色素瘤活检结果的关系。本研究将确定 真皮细胞外基质结构促进年龄相关的黑素瘤进展的机制， 其具有改善老年患者的生活质量的直接潜力。