对JAK2V617F突变所导致的骨髓增生性疾病的小鼠心脏肥大的研究
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摘要
骨髓增生性疾病（MPNs）是一种慢性恶性血液病，特点是一个或多个骨髓系统异常增生。这些疾病包括真性红细胞增多症（PV），血小板增多症（ET），和原发性骨髓纤维化（PMF）。PV病人三种类型血细胞均有增加，ET病人主要表现在血小板增多。由于骨髓增生异常，PMF病人会形成骨髓纤维化（瘢痕形状）。MPNs主要影响老年人群，平均发病年龄为55岁。目前没有针对该疾病的有效治疗方法。MPNs并发症主要有血栓形成、出血、心脏病发作、中风、骨髓组织髓样化生和急性白血病。由这些疾病引起的中风和心脏病发作通常是致命的。事实上，心血管事件代表PV和ET过程中发病率和死亡率的主要原因。然而，关于与MPNs相关的血液异常所导致的心脏改变，其原因目前尚不清楚。
JAK2V617F，酪氨酸激酶JAK2的突变形式，代表MPNs中主要的分子缺陷。在超过95%的PV和超过50%的ET和PMF病例中可发现该现象。在早期研究中，我们的合作单位美国俄克拉荷马大学健康科学中心病理实验室利用vav-1助催化剂，在造血系统中促进转基因表达，培养了JAK2V617F转基因小鼠。JAK2V617F转基因小鼠表现出与MPNs相似的表型，红细胞、血小板和白细胞的增多。因此这些小鼠可做为研究MPNs和相关并发症的模型系统。在目前的研究中，我们在这些小鼠身上通过MPNs表型的发展来研究心脏的变化和重构。
材料和方法
动物
用前面所述方法形成的JAK2V617F转基因小鼠。vav-1催化剂控制下的JAK2V617F转基因，其中vav-1催化剂只有在造血细胞里才能驱动基因表达。这些小鼠是与C57BL/6野生型小鼠十代杂交之后的后代。本研究中使用了携带纯合子A组的JAK2V617F转基因小鼠。这些小鼠携带26个JAK2V617F转基因复制物。野生型C57BL/6是从杰克逊实验室购买的，与没有进行转基因的JAK2V617F转基因小鼠的同胞一起做为对照组小鼠。至少有雌雄均匀的十只小鼠被用于对照组或实验组。所有的动物都被安置在通风的笼子里，处于标准的环境下。本研究严格遵守美国国立卫生研究院编订的实验动物护理和使用指南。
鼠组织的收集、固定和切片
通过使小鼠吸入异氟烷使其陷入深度麻醉状态进行测量。通过移除眼球来进行末梢血液采集。之后将心脏移除，去除血迹之后测量。紧接着将其固定在浓度10%的与室内温度相同的中性福尔马林中一夜，之后以石蜡封住。组织切片是从各种部位切下来的。
组织化学染色，图像采集和数字量化
将组织切片脱蜡，之后按照标准程序使用Sigma-Aldrich试剂进行H&E染色，马松三色染色，网状染色和甲苯胺蓝色染色。使用奥林巴斯BX-51直立显微镜配备萤石物镜观察目标切片。使用DP71数码相机获取图像，DP-BSW-V3.1作为摄像机控制软件（奥林巴斯），用AdobePhotoshop软件进行处理。组织化学染色图像的量化是用NIH Image程序完成的。每个小组至少分析十只小鼠的心脏部分。测量每个心脏的左心室钝缘厚度，肌小梁除外。随机选择心肌细胞上有H&E染色域的横截面测量评估心肌细胞大小。
总RNA提取和R-T PCR分析
使用RNeasy Mini Kit从小鼠组织中提取总RNAs，使用Qiagen的逆转录量化技术将等量的总RNAs合成单链cDNA。R-T PCR与绿色荧光定量PCR试剂（Bio-Rad）、小鼠內生的Jak2引物、转基因JAK2V617F、之前提到过的鼠磷酸甘油醛脱氢酶（GAPDH）能共同作用。
结果：
1、JAK2V617F转基因小鼠表现出心脏肥大
在40-45周期间，JAK2V617F转基因小鼠的心脏几乎比对照组小鼠的心脏重30%。小鼠在五周大时，心脏质量方面没有区别，在低倍镜下，用显微镜检查H&E染色部分，在JAK2V617F转基因小鼠身上有明显的左心室壁增厚。在高倍镜下继续检查发现心肌细胞体积增大。平均来讲，转基因小鼠的细胞面积比对照组小鼠大30%。
2、JAK2V617F转基因小鼠心脏中纤维化的发生
对JAK2V617F转基因小鼠心脏进行马松三色染色，可观察到转基因小鼠心脏左心室有突出的胶原纤维。在组织间隙和血管周围都有纤维化现象。与此相反，对照组小鼠相同区域的三色染色则是边缘化的。进一步利用网状染色研究III型胶原蛋白，转基因小鼠组织间隙的网状纤维集中，厚度很厚。对照组小鼠的心脏中细胞连接处的网状染色很明显，但是薄很多。
3、JAK2V617F转基因小鼠心脏中出现冠状动脉血栓及炎症细胞侵润
对20组年龄在40-70周的JAK2V617F转基因小鼠及20组对照组小鼠的超过5组H&E染色心脏切片进行进一步的检查。发现4只转基因小鼠的冠状动脉出现血栓症，但对照组中没有一只小鼠出现血栓症。我们还发现5只JAK2V617F转基因小鼠的心脏切片呈现炎症细胞侵润，而对照组小鼠中并没有出现。三色染色与网状染色可以在影响区域内显示出胶原蛋白纤维化。甲苯胺蓝染色可以在侵润炎症细胞中显示出肥大细胞。
4、实时PCR分析可以揭示JAK2V617F转基因小鼠心脏的最小表达
为了确定心脏中内因性Jak2和转基因JAK2V617F的表现，我们用特异引物来进行PCR实时分析这些基因在造血组织中的表现[3]。包括骨髓，周边血液和脾脏都用来作比对。心脏中转基因JAK2V617F的表现要弱的很多，大约是造血组织中内因性小鼠Jak2表达水平的1/200。此数据体现出小鼠心脏细胞中JAK2V617F转基因的最小表现。虽然我们不能完全排除心肌细胞中JAK2V617F表现能够导致心脏肥大，但这种可能性也相当之小。
结论：
我们已经表明小鼠体内JAK2V617F突变引发的骨髓增生性疾病（MPNs）能进一步导致心脏肥大。这对我们了解MPNs患者的并发症具有重大意义。我们的数据帮助解释了MPNs患者发病率和死亡率的原因，即心血管事件。我们进一步证明了，JAK2V617F转基因小鼠的心脏显然是发生了心肌纤维化，不仅涉及I型胶原蛋白，还涉及到网状纤维。JAK2V617F转基因小鼠将恶性血液病与心脏病联系起来，是研究心血管疾病和血液紊乱的独特模型系统。
Myeloproliferative neoplasms (MPNs) are chronic hematopoieticmalignancies characterized by abnormal amplification of one or moremyeloid lineages. These diseases include polycythemia vera (PV),essential thrombocythemia (ET), and primary myelofibrosis (PMF). PVpatients have increased production of all three types of blood cells,whereas ET patients mainly show elevations of platelets. Patients withPMF develop fibrous (scar-like) tissues in the bone marrow as a result ofabnormal myeloid proliferation. MPNs mainly affect older people with anaverage onset of55years. So far, there is no effective cure for the diseases.Complications associated with MPNs include thrombosis, hemorrhage,heart attacks, strokes, myeloid metaplasia, and acute leukemia. Strokesand heart attacks caused by these diseases are usually fatal. In fact,cardiovascular events represent the leading cause of morbidity andmortality in the course of PV and ET. However, how the bloodabnormality associated with MPNs leads to cardiac changes is not wellunderstood.
JAK2V617F, a mutant form of tyrosine kinase JAK2, represents amajor molecular defect in MPNs. It is found in over95%of PV and over50%of ET and PMF cases. In early studies, we have generatedJAK2V617F transgenic mice by using the vav-1promoter which drivestransgene expressions in the hematopoietic system. JAK2V617Ftransgenic mice display MPN-like phenotypes with increased levels of redblood cells, platelets, and white blood cells. These mice thus represent amodel system to study MPNs and associated complications. In the present study, we investigated cardiac changes and remodeling during the courseof MPN phenotype development in these mice.
Materials and methods
Animals
JAK2V617F transgenic mice were generated as previously described.The JAK2V617F transgene is under the control of the vav-1promoterwhich drives gene expression only in hematopoietic cells. These mice havebeen crossed with wild type C57BL/6mice for over10generations.Homozygous line A JAK2V617F mice were used in this study. These micecarry26copies of the JAK2V617F transgene. Wild type C57BL/6werepurchased from The Jackson Laboratory and used as control together withnon-transgenic siblings of JAK2V617F transgenic mice. At least10micewith about equal male and female representations were used for eachcontrol or experimental group. Animals were housed in ventilated cagesunder standard conditions. This study was carried out in strict accordancewith the recommendations in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health.
Mouse tissue collection, fixation, and sectioning
Mice were weighed and put under deep anesthesia through inhalationof isoflurane. Terminal blood collection was performed by removing theeyeball from the socket. Hearts were then removed, blotted free of blood,and weighed. This is followed by fixation in10%neutral bufferedformalin overnight at room temperature and subsequent embedding inparaffin. Tissue sections (5μm) were cut from various positions.
Histochemical staining, image acquisition, and digitalquantification
Tissue sections were deparaffinized and then subjected to H&E,Masson’s trichrome, reticulin, and toluidine blue staining by usingreagents and kits from Sigma-Aldrich following standard protocols. Slideswere viewed with an Olympus BX-51upright microscope equipped withU Plan Fluorite objectives. Images were acquired using a DP71digitalcamera with the DP-BSW-V3.1camera control software (Olympus) andwere processed with the Adobe Photoshop software. Quantification ofhistochemical stain images was done by using the NIH ImageJ program.Cardiac sections from at least10mice per group were analyzed. Thethickness at the obtuse margin of the left ventricle of each heart wasmeasured, excluding trabeculations. Cardiomyocyte size was assessed bymeasuring cross-sectional area of cardiomyocytes from H&E-stainedfields randomly selected.
Total RNA isolation and real time PCR analysis
Total RNAs were isolated from mouse tissues by using the RNeasyMini kit (Qiagen), and single strand cDNAs were synthesized with equalamounts of total RNAs by using the QuantiTect reverse transcription kitfrom Qiagen. Real time PCR was performed with iQ SYBR GreenSupermix (Bio-Rad) and primers specific for endogenous mouse Jak2,transgenic human JAK2V617F, and mouse glyceral-dehyde-3-phosphatedehydrogenase (GAPDH) as previously described.
Results
1.JAK2V617F transgenic mice displayed cardiomegaly
We employed mice of40–45weeks to characterize the cardiomegaly further. Microscopic examinations of hematoxylin and eosin(H&E)-stained cardiac sections under low magnification revealedsignificant thickening of the left ventricular wall in the JAK2V617Ftransgenic mice (P <0.01). Further exanimation under high magnificationdemonstrated enlarged cardiomyocytes (P <0.001). On average, the cellsfrom the transgenic mice were30%bigger in area than the control.
2. JAK2V617F transgenic mice developed fibrosis in the heart
we performed Masson’s trichrome staining, a commonly usedmethod to detect collagen fibers.As indicated by the bright blue staining,prominent collagen fibrosis was observed in the left ventricle of transgenicmouse hearts. The fibrosis occurred in both interstitial and perivascularregions. In contrast, trichrome staining in the cor respondent regions ofcontrol mouse hearts was marginal.We further employed reticulin stainingto investigate type III collagen. veryintensive and thick interstitial reticulinfibers were observed with the transgenic mice. Reticulin staining in thecell-cell junction of control mouse hearts was also apparent but were muchthinner.
3.Coronary artery thrombosis and inflammatory cell infiltrationwere found in the heart of JAK2V617F transgenic mice
We further examined at least5selected H&E-stained cardiac sectionsfrom each of20control and20JAK2V617F transgenic mice of40–70weeks of age. We found occurrence of thrombosis in the coronary arteryof4transgenic, but not a single control mice. We also noticed that cardiacsections from5JAK2V617F transgenic mice but not at all from controlmice had infiltration of inflammatory cells. Trichrome and reticulinstaining revealed collagen fibrosis in the affected regions, toluidine blue staining showed that mast cells were among the infiltrated inflammatorycells.
4. Real time PCR analyses revealed a minimal expression oftransgenic JAK2V167F in the mouse heart
To determine the expression of endogenous Jak2and transgenicJAK2V617F in the heart, we performed real time PCR analysis withspecific primers as described previously. Expression of these genes inhematopoietic tissues including bone marrow, peripheral blood, andspleen were included for comparison. However, expression of transgenicJAK2V617F in the heart was much lower, about200-fold below the levelof mouse Jak2in the heart or of JAKV617F in the hematopoietic tissues.The data thus demonstrate a minimal expression of JAK2V617F transgenein mouse heart cells. Although we cannot totally rule out the contributionof JAK2V617F expression in cardiomyocytes to the development ofcardiac hypertrophy phenotypes, the possibility for this is very low.
Conclusions
We have demonstrated that JAK2V617F-induced MPNs can furtherlead to cardiac hypertrophy in mice. This has major implications for ourunderstanding of complications that occur in MPN patients. Our data helpto explain the high morbidity and mortality of MPN patients due tocardiovascular events. We have further demonstrated that the cardiacremodeling in JAK2V617F transgenic mice is manifest in fibrosisinvolving not only type I collagen but also type III reticulin fibers. Bylinking a hematological malignancy with heart diseases, JAK2V617Ftransgenic mice thus represent a unique model system to studycardiovascular diseases as well as blood disorders.
JAK2V617F，酪氨酸激酶JAK2的突变形式，代表MPNs中主要的分子缺陷。在超过95%的PV和超过50%的ET和PMF病例中可发现该现象。在早期研究中，我们的合作单位美国俄克拉荷马大学健康科学中心病理实验室利用vav-1助催化剂，在造血系统中促进转基因表达，培养了JAK2V617F转基因小鼠。JAK2V617F转基因小鼠表现出与MPNs相似的表型，红细胞、血小板和白细胞的增多。因此这些小鼠可做为研究MPNs和相关并发症的模型系统。在目前的研究中，我们在这些小鼠身上通过MPNs表型的发展来研究心脏的变化和重构。
材料和方法
动物
用前面所述方法形成的JAK2V617F转基因小鼠。vav-1催化剂控制下的JAK2V617F转基因，其中vav-1催化剂只有在造血细胞里才能驱动基因表达。这些小鼠是与C57BL/6野生型小鼠十代杂交之后的后代。本研究中使用了携带纯合子A组的JAK2V617F转基因小鼠。这些小鼠携带26个JAK2V617F转基因复制物。野生型C57BL/6是从杰克逊实验室购买的，与没有进行转基因的JAK2V617F转基因小鼠的同胞一起做为对照组小鼠。至少有雌雄均匀的十只小鼠被用于对照组或实验组。所有的动物都被安置在通风的笼子里，处于标准的环境下。本研究严格遵守美国国立卫生研究院编订的实验动物护理和使用指南。
鼠组织的收集、固定和切片
通过使小鼠吸入异氟烷使其陷入深度麻醉状态进行测量。通过移除眼球来进行末梢血液采集。之后将心脏移除，去除血迹之后测量。紧接着将其固定在浓度10%的与室内温度相同的中性福尔马林中一夜，之后以石蜡封住。组织切片是从各种部位切下来的。
组织化学染色，图像采集和数字量化
将组织切片脱蜡，之后按照标准程序使用Sigma-Aldrich试剂进行H&E染色，马松三色染色，网状染色和甲苯胺蓝色染色。使用奥林巴斯BX-51直立显微镜配备萤石物镜观察目标切片。使用DP71数码相机获取图像，DP-BSW-V3.1作为摄像机控制软件（奥林巴斯），用AdobePhotoshop软件进行处理。组织化学染色图像的量化是用NIH Image程序完成的。每个小组至少分析十只小鼠的心脏部分。测量每个心脏的左心室钝缘厚度，肌小梁除外。随机选择心肌细胞上有H&E染色域的横截面测量评估心肌细胞大小。
总RNA提取和R-T PCR分析
使用RNeasy Mini Kit从小鼠组织中提取总RNAs，使用Qiagen的逆转录量化技术将等量的总RNAs合成单链cDNA。R-T PCR与绿色荧光定量PCR试剂（Bio-Rad）、小鼠內生的Jak2引物、转基因JAK2V617F、之前提到过的鼠磷酸甘油醛脱氢酶（GAPDH）能共同作用。
结果：
1、JAK2V617F转基因小鼠表现出心脏肥大
在40-45周期间，JAK2V617F转基因小鼠的心脏几乎比对照组小鼠的心脏重30%。小鼠在五周大时，心脏质量方面没有区别，在低倍镜下，用显微镜检查H&E染色部分，在JAK2V617F转基因小鼠身上有明显的左心室壁增厚。在高倍镜下继续检查发现心肌细胞体积增大。平均来讲，转基因小鼠的细胞面积比对照组小鼠大30%。
2、JAK2V617F转基因小鼠心脏中纤维化的发生
对JAK2V617F转基因小鼠心脏进行马松三色染色，可观察到转基因小鼠心脏左心室有突出的胶原纤维。在组织间隙和血管周围都有纤维化现象。与此相反，对照组小鼠相同区域的三色染色则是边缘化的。进一步利用网状染色研究III型胶原蛋白，转基因小鼠组织间隙的网状纤维集中，厚度很厚。对照组小鼠的心脏中细胞连接处的网状染色很明显，但是薄很多。
3、JAK2V617F转基因小鼠心脏中出现冠状动脉血栓及炎症细胞侵润
对20组年龄在40-70周的JAK2V617F转基因小鼠及20组对照组小鼠的超过5组H&E染色心脏切片进行进一步的检查。发现4只转基因小鼠的冠状动脉出现血栓症，但对照组中没有一只小鼠出现血栓症。我们还发现5只JAK2V617F转基因小鼠的心脏切片呈现炎症细胞侵润，而对照组小鼠中并没有出现。三色染色与网状染色可以在影响区域内显示出胶原蛋白纤维化。甲苯胺蓝染色可以在侵润炎症细胞中显示出肥大细胞。
4、实时PCR分析可以揭示JAK2V617F转基因小鼠心脏的最小表达
为了确定心脏中内因性Jak2和转基因JAK2V617F的表现，我们用特异引物来进行PCR实时分析这些基因在造血组织中的表现[3]。包括骨髓，周边血液和脾脏都用来作比对。心脏中转基因JAK2V617F的表现要弱的很多，大约是造血组织中内因性小鼠Jak2表达水平的1/200。此数据体现出小鼠心脏细胞中JAK2V617F转基因的最小表现。虽然我们不能完全排除心肌细胞中JAK2V617F表现能够导致心脏肥大，但这种可能性也相当之小。
结论：
我们已经表明小鼠体内JAK2V617F突变引发的骨髓增生性疾病（MPNs）能进一步导致心脏肥大。这对我们了解MPNs患者的并发症具有重大意义。我们的数据帮助解释了MPNs患者发病率和死亡率的原因，即心血管事件。我们进一步证明了，JAK2V617F转基因小鼠的心脏显然是发生了心肌纤维化，不仅涉及I型胶原蛋白，还涉及到网状纤维。JAK2V617F转基因小鼠将恶性血液病与心脏病联系起来，是研究心血管疾病和血液紊乱的独特模型系统。
Myeloproliferative neoplasms (MPNs) are chronic hematopoieticmalignancies characterized by abnormal amplification of one or moremyeloid lineages. These diseases include polycythemia vera (PV),essential thrombocythemia (ET), and primary myelofibrosis (PMF). PVpatients have increased production of all three types of blood cells,whereas ET patients mainly show elevations of platelets. Patients withPMF develop fibrous (scar-like) tissues in the bone marrow as a result ofabnormal myeloid proliferation. MPNs mainly affect older people with anaverage onset of55years. So far, there is no effective cure for the diseases.Complications associated with MPNs include thrombosis, hemorrhage,heart attacks, strokes, myeloid metaplasia, and acute leukemia. Strokesand heart attacks caused by these diseases are usually fatal. In fact,cardiovascular events represent the leading cause of morbidity andmortality in the course of PV and ET. However, how the bloodabnormality associated with MPNs leads to cardiac changes is not wellunderstood.
JAK2V617F, a mutant form of tyrosine kinase JAK2, represents amajor molecular defect in MPNs. It is found in over95%of PV and over50%of ET and PMF cases. In early studies, we have generatedJAK2V617F transgenic mice by using the vav-1promoter which drivestransgene expressions in the hematopoietic system. JAK2V617Ftransgenic mice display MPN-like phenotypes with increased levels of redblood cells, platelets, and white blood cells. These mice thus represent amodel system to study MPNs and associated complications. In the present study, we investigated cardiac changes and remodeling during the courseof MPN phenotype development in these mice.
Materials and methods
Animals
JAK2V617F transgenic mice were generated as previously described.The JAK2V617F transgene is under the control of the vav-1promoterwhich drives gene expression only in hematopoietic cells. These mice havebeen crossed with wild type C57BL/6mice for over10generations.Homozygous line A JAK2V617F mice were used in this study. These micecarry26copies of the JAK2V617F transgene. Wild type C57BL/6werepurchased from The Jackson Laboratory and used as control together withnon-transgenic siblings of JAK2V617F transgenic mice. At least10micewith about equal male and female representations were used for eachcontrol or experimental group. Animals were housed in ventilated cagesunder standard conditions. This study was carried out in strict accordancewith the recommendations in the Guide for the Care and Use of LaboratoryAnimals of the National Institutes of Health.
Mouse tissue collection, fixation, and sectioning
Mice were weighed and put under deep anesthesia through inhalationof isoflurane. Terminal blood collection was performed by removing theeyeball from the socket. Hearts were then removed, blotted free of blood,and weighed. This is followed by fixation in10%neutral bufferedformalin overnight at room temperature and subsequent embedding inparaffin. Tissue sections (5μm) were cut from various positions.
Histochemical staining, image acquisition, and digitalquantification
Tissue sections were deparaffinized and then subjected to H&E,Masson’s trichrome, reticulin, and toluidine blue staining by usingreagents and kits from Sigma-Aldrich following standard protocols. Slideswere viewed with an Olympus BX-51upright microscope equipped withU Plan Fluorite objectives. Images were acquired using a DP71digitalcamera with the DP-BSW-V3.1camera control software (Olympus) andwere processed with the Adobe Photoshop software. Quantification ofhistochemical stain images was done by using the NIH ImageJ program.Cardiac sections from at least10mice per group were analyzed. Thethickness at the obtuse margin of the left ventricle of each heart wasmeasured, excluding trabeculations. Cardiomyocyte size was assessed bymeasuring cross-sectional area of cardiomyocytes from H&E-stainedfields randomly selected.
Total RNA isolation and real time PCR analysis
Total RNAs were isolated from mouse tissues by using the RNeasyMini kit (Qiagen), and single strand cDNAs were synthesized with equalamounts of total RNAs by using the QuantiTect reverse transcription kitfrom Qiagen. Real time PCR was performed with iQ SYBR GreenSupermix (Bio-Rad) and primers specific for endogenous mouse Jak2,transgenic human JAK2V617F, and mouse glyceral-dehyde-3-phosphatedehydrogenase (GAPDH) as previously described.
Results
1.JAK2V617F transgenic mice displayed cardiomegaly
We employed mice of40–45weeks to characterize the cardiomegaly further. Microscopic examinations of hematoxylin and eosin(H&E)-stained cardiac sections under low magnification revealedsignificant thickening of the left ventricular wall in the JAK2V617Ftransgenic mice (P <0.01). Further exanimation under high magnificationdemonstrated enlarged cardiomyocytes (P <0.001). On average, the cellsfrom the transgenic mice were30%bigger in area than the control.
2. JAK2V617F transgenic mice developed fibrosis in the heart
we performed Masson’s trichrome staining, a commonly usedmethod to detect collagen fibers.As indicated by the bright blue staining,prominent collagen fibrosis was observed in the left ventricle of transgenicmouse hearts. The fibrosis occurred in both interstitial and perivascularregions. In contrast, trichrome staining in the cor respondent regions ofcontrol mouse hearts was marginal.We further employed reticulin stainingto investigate type III collagen. veryintensive and thick interstitial reticulinfibers were observed with the transgenic mice. Reticulin staining in thecell-cell junction of control mouse hearts was also apparent but were muchthinner.
3.Coronary artery thrombosis and inflammatory cell infiltrationwere found in the heart of JAK2V617F transgenic mice
We further examined at least5selected H&E-stained cardiac sectionsfrom each of20control and20JAK2V617F transgenic mice of40–70weeks of age. We found occurrence of thrombosis in the coronary arteryof4transgenic, but not a single control mice. We also noticed that cardiacsections from5JAK2V617F transgenic mice but not at all from controlmice had infiltration of inflammatory cells. Trichrome and reticulinstaining revealed collagen fibrosis in the affected regions, toluidine blue staining showed that mast cells were among the infiltrated inflammatorycells.
4. Real time PCR analyses revealed a minimal expression oftransgenic JAK2V167F in the mouse heart
To determine the expression of endogenous Jak2and transgenicJAK2V617F in the heart, we performed real time PCR analysis withspecific primers as described previously. Expression of these genes inhematopoietic tissues including bone marrow, peripheral blood, andspleen were included for comparison. However, expression of transgenicJAK2V617F in the heart was much lower, about200-fold below the levelof mouse Jak2in the heart or of JAKV617F in the hematopoietic tissues.The data thus demonstrate a minimal expression of JAK2V617F transgenein mouse heart cells. Although we cannot totally rule out the contributionof JAK2V617F expression in cardiomyocytes to the development ofcardiac hypertrophy phenotypes, the possibility for this is very low.
Conclusions
We have demonstrated that JAK2V617F-induced MPNs can furtherlead to cardiac hypertrophy in mice. This has major implications for ourunderstanding of complications that occur in MPN patients. Our data helpto explain the high morbidity and mortality of MPN patients due tocardiovascular events. We have further demonstrated that the cardiacremodeling in JAK2V617F transgenic mice is manifest in fibrosisinvolving not only type I collagen but also type III reticulin fibers. Bylinking a hematological malignancy with heart diseases, JAK2V617Ftransgenic mice thus represent a unique model system to studycardiovascular diseases as well as blood disorders.
引文
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