Transmembrane Stem Factor Nanodiscs Enhanced Revascularization in a Hind Limb Ischemia Model in Diabetic, Hyperlipidemic Rabbits

Therapies to revascularize ischemic tissue have long been a goal for the treatment of vascular disease and other disorders. Therapies using stem cell factor (SCF), also known as a c-Kit ligand, had great promise for treating ischemia for myocardial infarct and stroke, however clinical development for SCF was stopped due to toxic side effects including mast cell activation in patients. We recently developed a novel therapy using a transmembrane form of SCF (tmSCF) delivered in lipid nanodiscs. In previous studies, we demonstrated tmSCF nanodiscs were able to induce revascularization of ischemia limbs in mice and did not activate mast cells. To advance this therapeutic towards clinical application, we tested this therapy in an advanced model of hindlimb ischemia in rabbits with hyperlipidemia and diabetes. This model has therapeutic resistance to angiogenic therapies and maintains long term deficits in recovery from ischemic injury. We treated rabbits with local treatment with tmSCF nanodiscs or control solution delivered locally from an alginate gel delivered into the ischemic limb of the rabbits. After eight weeks, we found significantly higher vascularity in the tmSCF nanodisc-treated group in comparison to alginate treated control as quantified through angiography. Histological analysis also showed a significantly higher number of small and large blood vessels in the ischemic muscles of the tmSCF nanodisc treated group. Importantly, we did not observe inflammation or mast cell activation in the rabbits. Overall, this study supports the therapeutic potential of tmSCF nanodiscs for treating peripheral ischemia.


Introduction
Diabetes mellitus affects approximately 350 million people, leading to the death of an estimated 4.6 million people in the world per year 1,2 . Diabetes leads to a disturbance of the blood vessel by promoting vascular in ammation and endothelial cell dysfunction 3,4 . These abnormalities increase the severity of vascular disease in diabetic patients 5 . As a complication of diabetes, 30 to 40 percent of patients age 50 and older develop peripheral artery disease (PAD) 6 . Severe PAD increases the risk of nonhealing ulcers, pain from intermittent claudication, and worst case for limb amputation. Current standard cares for PAD include physical therapy, medication, and surgical revascularization. Surgical bypass is an important treatment option for sever PAD, however many patients especially elderly patients are not eligible because of their diffusive arterial occlusions, no suitable veins for grafting and comorbidity 7 .
Therapeutic angiogenesis by growth factors or growth factor genes is an appealing strategy to treat ischemia in this context but has been di cult to translate in the clinical setting. Many growth factors/gene therapies have shown promise in preclinical studies for ischemia only to show disappointing results in clinical trials in human patients [8][9][10] .
Stem cell factor (SCF) is a hematopoietic cytokine that communicates via the c-Kit receptor (CD117) and is also known as Kit ligand, Steel factor, or mast cell growth factor 11 . Stem cell factor is produced in cells as a transmembrane protein, which is then enzymatically processed into soluble SCF or a shorter version without the cleavable domain, that remains membrane-bound as transmembrane SCF (tmSCF) 12 . The activation of c-Kit through SCF signaling is crucial for maintaining hematopoietic stem cells (HSCs) and other progenitor cells in the bone marrow 13,14 . There are many potential therapeutic applications for SCF, including the enhancement of survival and expansion of HSCs after radiation exposure 15,16 , providing neuroprotection following a stroke [17][18][19] , and aiding the heart's recovery after myocardial infarction 20 . In addition, SCF plays a crucial role in controlling mast cell development and activation 21,22 . Unfortunately, clinical and animal studies of SCF treatment have found induction of mast cell activation and anaphylaxis, signi cantly restricting its potential for therapeutic use [22][23][24][25][26] . We recently found that tmSCF delivered in proteoliposomes or lipid nanodiscs was effective in inducing revascularization of ischemic limbs in diabetic and wild type mice but did not lead to mast cell activation 27 . Thus, tmSCF delivered in nanocarriers may provide therapeutic bene ts of soluble SCF without the toxic side effects that limited its usefulness as a therapeutic.
While our previous studies have supported the potential of tmSCF nanodiscs as therapies for ischemia, large animal studies are needed to demonstrate e cacy to support further studies in human trials. For peripheral ischemia, rabbits are typically used as the precursor to human studies for treating PAD due the well-developed musculature of the lower limb. Our group recently developed an optimized rabbit model of hindlimb ischemia that includes hyperlipidemia and diabetes 28,29 . A major advantage of this model is that it exhibits resistance to angiogenic therapies and longer-term ischemia, similar to human patients, in contrast to health rabbit models that revascularize more quickly and have been found to correlate poorly to outcomes in clinical trials. In this study, we used this advanced rabbit model of limb ischemia to evaluate the e cacy of tmSCF nanodiscs in treating peripheral ischemia. Overall, our results demonstrate that tmSCF nanodiscs improve revascularization in ischemia compared to alginate gel control in a large animal without signi cant in ammatory reaction.

Methods
Preparation of tmSCF Nanodiscs. A solution of 50 mM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was created in chloroform and then evaporated to make a thin lm. The POPC was resuspended in a solution of 100 mM sodium deoxycholate using multiple cycles of sonication. The MSP protein (5 mg/mL MSP1D1) was then added to phospholipid solution, and the detergent concentration was adjusted to 40 mM. This construct was then incubated for 15 min at 4°C. To solubilize the membrane protein, tmSCF was incubated in the n-octyl-β-D-glucopyranoside (1% w/v) for 15 min at 4°C. In the separate tube, a solution was created of 1 µg/ml, 1% n-octyl-β-D-glucopyranoside (1% w/v) and 40 mM sodium deoxycholate. The POPC and tmSCF solution were combined for 1 hour at 4°C. The combined solution was then dialysed overnight use a 300K MWCO dialysis membrane. Biobeads (SM-2; Bio-Rad Laboratories, Inc) were then used to remove any residual detergent (Supplemental Fig. 1).
Preparation of Alginate Gels and Crosslinking. Sterile alginate powder (Sigma) was added to sterile saline to create a 2% w/v solution. The nal concentration of tmSCF nanodiscs was adjusted to 50 µg/ml in this 2% alginate solution. To prepare the crosslinker, calcium sulfate was added to sterile saline to create 0.2% w/v solution. Both the alginate solution and the crosslinker were taken up into a 1 ml syringe just before injection (100 µl of each solution for 200 µl total per injection). with NIH guidelines for animal care. The studies performed were in accordance with ARRIVE guidelines. New Zealand rabbits were transitioned from standard alfalfa chow to a 0.1% cholesterol diet over the course of ve days. After two weeks on the 0.1% cholesterol diet, rabbits were induced with diabetes using an intravenous alloxan injection. After sedating the rabbits, a bassline blood glucose measurement was attained. Alloxan (100 mg/kg) was injected through an IV into the rabbit at ow rate of 1 ml/min for eight minutes using a syringe pump. Blood glucose levels were monitored closely for 12 hours after the injection. A successful induction of diabetes was determined if the rabbit's blood glucose level remained over 150 mg/dl prior to insulin administration.

Induction of Diabetes in
Hind limb Ischemia Surgery and Treatments. To induce ischemia in the hind limb of New Zealand rabbits, a longitudinal incision was made in the skin over the femoral artery. The femoral artery was exposed using blunt dissection. One percent lidocaine was applied to the area to reduce nerve irritation and promote vasodilation. Continued blunt dissection was used to expose the entire length of the femoral artery and branches including the inferior epigastric, deep femoral, lateral circum ex, and super cial epigastric arteries. The tissue was kept moistened with saline to avoid damage. The femoral artery was then carefully separated from vein and nerve. The femoral artery was then ligated with 4.0 silk sutures, cut, and excised. Two weeks after hind limb ischemia surgery, ten syringes containing 100 µl of alginate with treatment and 100 µl of calcium sulfate crosslinker were prepared for intramuscular injection (200 µl total volume of injection). Treatments included tmSCF nanodiscs in alginate and an alginate-only control.
Angiography Quanti cation. Angiograms were quanti ed using grid analysis techniques with ImageJ Software. Brie y, brightness and contrast were adjusted to better visualize vessels. A grid overlay of 100 pixels per square was used for foot quanti cation and 750 pixels per square was used for the thigh vasculature quanti cation. The multi-point tool was used to count intersections between the grid and vessels. To access the carotid artery, an incision was made just lateral to the trachea. Blunt dissection was used to expose the carotid artery and separate it from the jugular vein and vagus nerve. Ligatures were placed at the proximal and distal ends of the carotid artery. The distal end was tied off and a ligaloop was placed at the proximal end. A wire insertion tool was then inserted into the artery. Using the tool, a guidewire was fed into the artery to the aortic bifurcation in the descending aorta. The insertion tool was then removed and a 3F pigtail angiographic catheter was placed over the wire and advanced 2 cm proximal to the aortic bifurcation. Nitroglycerine and lidocaine were given to increase vasodilation. Contrast media was injected through the catheter using an automated angiographic injector. Angiography was performed before femoral ligation, after femoral ligation, and before sacri ce at week 10.
Statistical Analysis. All results are shown as mean ± standard error of the mean. Comparisons between only two groups were performed using a 2-tailed Student's t-test. Differences were considered signi cant at p < 0.05. Multiple comparisons between groups were analyzed by 2-way ANOVA followed by a Tukey post-hoc test. A 2-tailed probability value < 0.05 was considered statistically signi cant.

Results
Transmembrane SCF nanodiscs enhance revascularization of the ischemic thigh muscles of diabetic, hyperlipidemic rabbits. Rabbits were given a high fat diet for four weeks and were induced to develop diabetes two weeks prior to surgery for inducing unilateral limb ischemia as described previously 28, 29 . Angiograms were taken before and immediately after the surgery to assess the induction of ischemia in the limbs (Supplemental Fig. 2). The rabbits were then allowed to recover for two weeks to avoid treatment during the acute healing phase of recovery 29 . The rabbits were given ten injections of alginate with or without the tmSCF nanodiscs and recovery was assess using angiography after 10 weeks ( Fig. 1A; Supplemental Fig. 2). Vessel grid intersection counts showed signi cant vasculature increase for the tmSCF nanodisc group compared to the alginate control (Fig. 1B). When ratioed to the contralateral control limb at week 10, the tmSCF nanodisc group signi cantly improved recovery compared to the alginate control (Fig. 1C). A ratio was also taken of the grid intersections counted in the ischemic thigh at week 10 to the intersections counted in the ischemic thigh before hind limb surgery. The results show a signi cant increase in vascularity for the tmSCF nanodisc group in comparison to the control group (Fig. 1D). A new vessel formation is indicated by tortuosity (corkscrew like morphology), which was also counted to assess angiogenic capacity of tmSCF nanodiscs. The results also showed a signi cant increase in tmSCF nanodisc group in comparison to control. (Fig. 1E).
Transmembrane SCF nanodiscs enhance revascularization of the ischemic calf and foot of diabetic, hyperlipidemic rabbits. Postoperative angiogram showed the most severe ischemia is the area of the foot and calf muscles (Supplemental Fig. 2). Ten weeks after the operation, robust vascular network formation were observed at the foot of the tmSCF nanodiscs treated group while almost few or no blood vessels was observed in the control group by angiography ( Fig. 2A). Quantitative analysis on vessel count showed a signi cantly higher blood vessel numbers in tmSCF nanodisc group in comparison to the control (Fig. 2B). Similarly, signi cantly higher ratio of vascular count at the ischemic calf and foot when ratioed to the contralateral control at week 10 (Fig. 2C). A ratio of vessel counts at the ischemic thigh at week 10 to the vessel counts before surgery also demonstrated the effectiveness of the tmSCF nanodisc treatment (Fig. 2D). Histological analysis con rmed increased vascularity in tmSCF nanodisc treated leg of rabbits with diabetes and hyperlipidemia. We next performed a histological analysis on biopsies from the ischemic muscle of the rabbits. PECAM immunostaining was used to count small and large vessels formation (Fig. 3A). Signi cantly more small vessels and large vessels were con rmed with tmSCF nanodisc treatment, indicating that tmSCF nanodiscs induced both angiogenesis and arteriogenesis in the rabbit ischemia model (Fig. 3B). Importantly, no signs of in ammation were was found on the H&E staining of thigh and calf muscle tissues.

Discussion
Stem cell factor is a therapeutic protein with many potential uses for treating disease. Unfortunately, its use has been severely limited by toxic effects that were observed in animal studies and human clinical trials [22][23][24][25][26] . Managing ischemia in diabetic individuals has presented continuing challenges in the clinical setting, particularly for those undergoing bypass operations or percutaneous procedures 30,31 where diabetic patients have worse outcomes and reduced bene ts from therapy and interventions. Our previous work demonstrated that tmSCF nanodiscs enhance revascularization in healthy and diabetic mice 32 . In this work, we evaluated the e cacy of tmSCF embedded in nanodiscs for therapeutic angiogenesis using an optimized rabbit model of hindlimb ischemia with diabetes and hyperlipidemia 28, 29 . Angiography of the thigh and calf muscle treated with tmSCF nanodiscs showed signi cantly higher numbers of functional blood vessels and new vessels. Histological analysis on PECAM staining and H&E staining also demonstrated signi cantly higher numbers of small and large vessel formations compared control, indicating that tmSCF nanodiscs support both angiogenesis and arteriogenesis. The ndings are particularly signi cant given the rabbit model's resistance to angiogenesis and longer-term ischemia 28, 29 . Finally, we did not observe any signs of mast cell activation on histological analysis, consistent with our prior studies in mice 27 .
Prior studies have explored the use of many treatments in rabbits and other large animals for treating peripheral ischemia and ischemia wounds 33,34 . A lack of uniformity in the techniques employed for performing and analyzing preclinical hindlimb ischemia models in rabbits makes it di cult to directly compare with other studies. In addition, the majority of prior studies have also used healthy rabbits, which have more rapid recovery from ischemia and responsiveness to angiogenic treatments. Overall, the improvement in revascularization observed in our research using a diseased rabbit model is comparable or superior to earlier studies that utilized healthy rabbit models of hindlimb ischemia to assess protein therapeutics 35-57 , cell therapies 58-62 , or gene therapies 55,63−75 . Consequently, this work supports that tmSCF nanodiscs would have potential as a therapeutic for ischemia, even in the context of therapeutic resistance as would be found in many human patients that have hyperlipidemia and diabetes.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.