Endothelial Cell-Specific Knockout of Meis1 Protects Ischemic Hindlimb Through Vascular Remodeling

Peripheral artery disease (PAD) affects more than 200 million people worldwide. PAD refers to illness due to a reduction or complete occlusion of blood flow in the artery, especially to the extremities in disease conditions, such as atherosclerosis or diabetes. Critical limb ischemia (CLI) is a severe form of PAD associated with high morbidity and mortality. Currently, no effective and permanent treatments are available for this disease. The current endovascular medications (e.g., angioplasty or stents) only relieve the clinical symptoms while the surgical therapies (e.g., bypass or endarterectomy) require grafting vessels from a healthy organ to the diseased limb of the patient. However, even with these therapeutic techniques, 30% of patients still undergo limb amputation within a year. Thus, understanding of disease mechanism and development of new therapeutic approaches are in urgent needs.

Meis1 (myeloid ecotropic viral integration site 1) gene belongs to the three-amino-acid loop extension subclass of homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. Up to date, little is known about the role of Meis1 in regulating vascular remodeling under ischemic condition. In this study, we aim to investigate the role and underlying mechanism of Meis1 in the regulation of arteriogenesis and angiogenesis using hindlimb ischemia model of transgenic neonatal mice. The long-term goal is to develop a new treatment for patients with PAD. Three separate but related studies were planned to complete the proposed research aims.

To better understand the role of Meis1, we reviewed, in the first chapter, all literature relevant to the recent advances of the Meis1 in normal hematopoiesis, vasculogenesis, and heart developments, which were mostly studied in zebrafish and mouse. Briefly, Meis1 is found to be highly expressed in the brain and retina in zebrafish and additional in the heart, nose, and limb in mouse during the very early developmental stage, and remains at a low level quickly after birth. Meis1 is necessary for both primitive and definitive hematopoiesis and required for posterior erythroid differentiation. The absence of Meis1 results in a severe reduction of the number of mature erythrocytes and weakens the heart beats in zebrafish. Meis1 deficiency mouse is dead as early as E11.5 due to the severe internal hemorrhage. In addition, Meis1 is essential in heart development. Knock-down of Meis1 can promote angiotensin II-induced cardiomyocytes (CMs) hypertrophy or CMs proliferation, which can be repressed by a transcription factor Tbx20. Meis1 appears to play a complicated role in the blood vessels. Although the major blood vessels are still normal when global deletion of Meis1, the intersegmental vessel cannot be formed in Meis1 morphants in the zebrafish, and the small vessels are either too narrow or form larger sinuses in Meis1 deficient mouse. The effects of Meis1 on the vascular network under normal and disease (ischemia) condition remain largely unknown, and the existing data in this field is limited.

In the second chapter, we developed a method protocol to identify mice of all ages, especially neonates that we faced methodological difficulties to easily and permanently label prior to our major experiments. In this study, single- or 2-color tattooing (ear, tail, or toe or combinations) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day (PND) 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than three weeks. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. Thus, tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals, which shows a new approach than any existing method to identify mouse at all ages, especially the neonatal pups used in the present study (Chapter 4).

Various formation of hindlimb ischemia with ligations of femoral artery or vein or both have been reported in the literature. The ischemic severity varies dependent on mouse strains and ligation methods. Due to the tiny body size of our experimental neonatal mice (PND2), it is technically challenging to separate the femoral artery from femoral vein without potential bleeding. In the third chapter, we aimed to explore a suitable surgical approach that can apply to neonatal mice. To this end, we compared the effects of femoral artery/vein (FAV) excision vs. femoral artery (FA) excision on hindlimb model using adult CD-1 mice. We showed during the 4-week period of blood reperfusion, no statistically significant differences were found between FAV and FA excision-induced ischemia regarding the reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell size. We conclude that FAV and FA excision in CD-1 mice generate a comparable severity of hindlimb ischemia. In other words, FAV ligation is no more severe than FA ligation. These findings provide valuable information for researchers when selecting ligation methods for their neonate hindlimb models. Based on these findings, we selected FAV ligation of hindlimb ischemia approach to study the function of Meis1 in vascular remodeling of neonatal mice. In the fourth chapter (the main part of my dissertation), we investigated the roles of Meis1 in regulating arteriogenesis and angiogenesis of neonatal mouse under the ischemic condition. To this end, endothelial cell-specific deletion of Meis1 was generated by cross-breeding Meis1flox/flox mice with Tie2-Cre mice. Wild-type (WT, Meis1f/f) and endothelial cell-specific knock-out (KO, Meis1ec-/-Tie2-Cre+) C57BL/6 mice at the age of PND2 were used. Under the anesthesia, the pups were subject to hindlimb ischemia by excising FAV. Laser Doppler Imager was used to measure the blood flow pre- and post-surgery up to 28 days. Toe necrosis, skeletal regeneration, and vascular distributions were examined at the end of experiments (PND28 post-ischemia). Surprisingly, during 4-week periods after ischemia, the blood flow ratios (ischemic vs. control limb) in KO mice significantly increased compared to WT on PND14 and PND28, suggesting the inhibitory effects of Meis1 on blood flow recovery under ischemic condition. Meanwhile, WT mice showed more severe necrotic limb (lower ratio of limb length and area, and higher necrotic scores at PND7) than those in the KO mice. Furthermore, significant increases in diameters of Dil-stained arterioles of the skin vessel and the vessels on the ligation site were observed in KO mice, indicating the enhanced arteriogenesis in KO mice. To investigate the underlying mechanism, RNA from the ischemia and control limb was extracted and q-PCR was used to study the potential genes involved in the mechanism. Casp3 and Casp8 were found downregulated showing less apoptosis in the KO mice. On the other hand, endothelial cells (ECs) were isolated from the lungs of 3-5 WT and KO neonates using CD31 Microbeads. CD31+ cells were plated and treated with 0, 0.5, and 1μM doxorubicin for 24 hours and analyzed with various assays. Meis1-KO ECs demonstrated higher cell viability and formed a higher number of vascular tubes than those in WT ECs following 0.5μM Dox treatment, presenting the potential ability of angiogenesis in KO-ECs. Furthermore, the increased viability in KO ECs may be due to the decreased expression or activities of Casp8 and Casp3.

In conclusion, my present studies have developed a new methodology to easily and permanently identify all mice at any ages. The insignificant differences between FAV and FA ligations suggest that a relative-easy surgical approach could be used to generate hindlimb ischemic model, which potentially reduces the cost, decreases the surgical time and prevents damage of femoral nerve from surgical tools. More importantly, by using transgenic mice, we found that Meis1-KO dramatically increased blood flow and protected the ischemic hindlimb through vascular remodeling. Obviously, the molecular and cellular mechanisms underlying the above beneficial effects appear complicated and likely to involve multiple cellular remodeling processes and molecular signaling pathways to enhance arteriogenesis and angiogenesis and/or reduce cellular apoptosis through Meis1-mediated pathways. Our study demonstrated that under ischemic condition, knockout of Meis1 increases expression of Hif1a, which then activates Agt or VEGF, thus enhances arteriogenesis or angiogenesis; In addition, knockout of Meis1 activates Ccnd1, which subsequently promotes regeneration of skeletal muscle, and reduces expression of Casp8 and Casp3, thus preventing limb tissue from ischemia-induced apoptosis. Our innovative findings offer great potential to ultimately lead to new drug discovery or therapeutic approaches for prevention or treatment of PAD.