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

转移性黑色素瘤的存活率只有23%。55岁以上的黑色素瘤患者 转移率低于低龄患者，独立于其他临床预后因素。我们的实验室有 研究表明，肿瘤微环境中年龄相关的变化足以驱动这种差异。在……里面 特别是，在衰老的真皮中，一种名为HAPLN1的分泌细胞外基质蛋白的丢失会增加 转移。HAPLN1的损失与基质分解相吻合，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壁垒 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血管内和转移。我的 初步研究表明，与老年小鼠相比，老年小鼠长出的黑色素瘤血管明显更多 敬年幼的老鼠。此外，这些血管显著降低了VE-钙粘附素的表达，这是一种 内皮细胞连接蛋白对维持血管完整性至关重要。然而，用药物治疗衰老的小鼠 重组HAPLN1足以挽救这些表型。VE-钙粘附素的表达受 正黏附受体/细胞外基质配体相互作用。因此，我们确定了两个 内皮细胞黏附受体在衰老的ECM中下调，其表达是 依赖于HAPLN1的存在。有趣的是，我们还发现，衰老的小鼠失去了胶原蛋白 包围着他们的血管，这些血管通常起到支持血管完整性和粘附性的作用 莱兰德。最后，我们已经证明，在体外，HAPLN1的存在维持了内皮屏障的功能。 它们的底物基质。我假设ECM的结构缺陷是由年龄相关性的丧失引起的 HAPLN1通过异常黏附受体/ECM相互作用损害血管完整性并增加 血源性渗入。为了研究这一假说，我将实现以下目标。目标1：确定 HAPLN1缺失降低血管完整性的机制。我会决定是否 受体/配体相互作用需要以HAPLN1依赖的方式调节VE-钙粘蛋白的表达 通过近端结扎实验和免疫荧光显微镜观察。我还将分析HAPLN1对 使用体外微流控平台检测血管完整性。目标2：确定HAPLN1对 黑色素瘤细胞在体内外的侵袭。我将评估HAPLN1对体外培养的贡献 黑色素瘤细胞的血管内“倒置”实验。我将评估甲型HAPLN1病毒S在 通过血管成像进行活体血管内注射。最后，我将分析HAPLN1、VE-钙粘蛋白和 人类黑色素瘤活检中的血管生成及其与年龄和预后的关系。这项研究将确定 真皮细胞外基质结构促进老年性黑色素瘤进展的机制 这对改善老年患者的预后具有直接的潜力。

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.