文献学习082--Bone Marrow Endothelial

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HSPCs reside in the bone marrow niche, which is formed by endothelial cells, mesenchymal stromal cells, macrophages, and osteoblasts.

Normal and Leukemic Stem Cell Niches: Insights and Therapeutic Opportunities

1. Diabetes Mellitus Increases Myelopoiesis

作者首先使用STZ连续（5d）注射构建了一型糖尿病小鼠模型，随后在4w, 8w, 12w, 16w时间点对小鼠外周血进行了流式检测，在16w时对小鼠骨髓也进行了流式检测，指标如下：

myeloid cells: CD19^low CD11b^high
monocytes: CD19^low CD11b^high CD115^high Ly6G^low
Ly- 6C^high monocytes: CD19^low CD11b^high CD115^high Ly6G^low Ly6C^high
neutrophils: CD19^low CD11b^high CD115^low Ly6G^high
(CD115就是CSF1R)

结果显示病程中小鼠外周血中myeloid cells，monocytes，Ly- 6C^high monocytes和neutrophils增加 (C)，16w时骨髓的这几种细胞也有增加的趋势 (E)。同时作者对骨髓中的下面三种细胞做了检测，发现GMP, LSK和HSCs显著增加 (G)。

granulocyte–macrophage progenitors: lineage^low c-Kit^high Sca1^low CD16/32^high CD34^high
Lin^-Sca-1⁺ c-Kit⁺ (LSK) cells: lineage^low c-Kit^high Sca1^high
hematopoietic stem cells (HSCs): lineage^low c-Kit^high Sca1^high CD48^low CD150^high

随后作者进行了5-bromo-2‘-deoxyuridine (BrdU) pulse chase experiment，2 weeks of BrdU pulse and 4 weeks of chase之后，作者检测了LSK和HSC的增殖情况，发现LSK和HSK的Brdu⁺细胞减少，indicating accelerated BrdU washout and enhanced proliferation。

二型糖尿病小鼠也是一样的表现。附件中作者对db鼠也做了检测，也是一样的表现。

2. Diabetes Mellitus Alters Bone Marrow Endothelial Cells

骨髓微环境对HSC的调控

Haematopoietic stem cell activity and interactions with the niche

Fig 3A： 为了探究myelopioesis的机制，作者首先分选了different niche cells（以下三种细胞）

内皮细胞：Lin^-CD45^-CD31^highSca-1^high
Mesenchemal Cell间充质干细胞：Lin^-CD45^-CD31^-Sca-1⁺CD51⁺
Osteoblasts成骨细胞：Lin^-CD45^-CD31^-Sca-1^-CD51⁺

Fig 3B： QPCR检测发现内皮细胞的Cxcl12表达下降（Cxcl12 is an important endothelial cell–derived niche factor that maintains HSC quiescence by interac- tion with its cognate receptor Cxcr4）
Fig 3C： 随后作者使用荧光检测了骨髓组织中 CD150⁺ HSC 和 CD31⁺ VE-cadherin⁺内皮细胞的共定位情况。
Fig 3D：结果显示在糖尿病小鼠骨髓中，HSC和内皮的距离更近。Together, these results indicate that the diabetic milieu affects endothelial cell–HSPC relations prominently.
Fig 3E：为了进一步明确糖尿病小鼠的骨髓内皮细胞变化，作者分选了糖尿病小鼠和正常小鼠的骨髓内皮细胞，进行了转录组测序。作者只得到了一个差异基因(Rabggtb)。但GSEA分析发现很多pathway出现了显著富集。而且富集到的pathway主要与炎症反应相关。
Fig 3F, G：展示了最显著富集的通路和其中的基因（炎症相关）。
Fig 3H, I：a gene set associated with obesity也在糖尿病小鼠中富集到。
Fig 3J, K：第二显著富集的通路是response to epidermal growth factor (EGF)

然后就定位到EGFR上面了？

Fig 3L：QPCR也显示骨髓内皮细胞EGFR表达上调
Fig 3M：流式显示骨髓内皮细胞EGFR磷酸化增加
Fig 3N：多种EGFR的配体包括EGF, TGF-a, 和 amphiregulin，可以活化Egfr tyro- sine kinase. 为了探究是哪种配体活化了EGFR，作者检测了对照和糖尿病小鼠血浆中这几个指标的水平，发现糖尿病小鼠血浆中amphiregulin显著上升。
Fig 3O：作者进一步探究了amphiregulin的来源，发现主要来自visceral adipose tissue compartment。

3. Endothelial Cell–Specific Egfr Deletion Enhances Myelopoiesis

随后作者构建了内皮特异性EGFR敲除鼠（Cdh5^Cre Egfr^fl/fl）。
Fig 4A, B：正常条件下，Cdh5^Cre Egfr^fl/fl与对照鼠的各种细胞比例没有差别（附件）。而高脂喂养下，Cdh5^Cre Egfr^fl/fl鼠的外周血髓系细胞显著增加。
Fig 4C, D：Cdh5^Cre Egfr^fl/fl鼠的骨髓LSK, GMP和HSC也显著增加。
Fig 4E, F：Cdh5^Cre Egfr^fl/fl鼠的HSC的增殖也增加
Fig 4G：为了探究是哪种 niche factors 介导了增强的 progenitor cell proliferation, 作者从骨髓中分离了diabetic Cdh5^Cre Egfr^fl/fl 和 control 鼠的内皮细胞，通过QPCR检测了most common niche factors，发现其他因子没有显著变化，angiopoietin-1显著下调。

Taken together, these data imply that increased Egfr signaling in bone marrow endothelial cells of diabetic mice represents an anti-inflammatory pathway that is not active in the steady state but counters the diabetes mellitus–associated increased leukocyte output of the marrow when mice are fed a high-fat diet. Of note, other conditions may also alter the same or different Egfr ligands, perhaps with diverging effects on hematopoiesis.

4. Disrupted Endothelial EGF Signaling Impairs Wound Healing in Diabetes Mellitus

也是表型，检测了diabetic Cdh5^Cre Egfr^fl/fl 小鼠的伤口愈合情况，发现愈合更快

5. Disrupted Endothelial EGF Signaling Accelerates Atherosclerosis

Accelerated development of coronary artery disease frequently complicates diabetes mellitus. To determine the functional relevance of enhanced myelopoiesis in endothelial Egfr-deficient mice in the setting of vascular inflammation, we used AAV2/8-driven PCSK9 production to induce atherosclerosis. 发现diabetic Cdh5^Cre Egfr^fl/fl 小鼠动脉粥样硬化情况更重。

6. Enhanced EGFR Signaling in Human Diabetes Mellitus

最后在人的样本做了一下验证。