Differences in Biologic Drug Effects and Distal Particulate Embolization in Three Paclitaxel-Coated Balloons for Femoropopliteal Lesions in a Rabbit Model.

BACKGROUND: A recent meta-analysis of randomized control trials demonstrated a significantly higher risk of major amputation in patients treated with drug-coated balloons (DCBs) compared with standard treatment, especially in high-dose paclitaxel-coated DCBs. Distal particulate embolization after DCB use was considered a potential cause of the higher incidence of major amputation. The current study aimed to histologically and biologically compare biologic drug effect and distal particulate embolization in 3 DCBs (a high-dose paclitaxel-coated DCB [IN.PACT Admiral] and 2 low-dose paclitaxel-coated DCBs [Ranger and Lutonix]). METHODS AND RESULTS: The DCBs were inflated in the healthy descending aortas of 18 rabbits, followed by euthanasia 28 days after the procedure. The treated descending aorta and distal skeletal muscles were histopathologically evaluated, and paclitaxel concentrations were measured. The paclitaxel concentration of the treated lesion was highest for Ranger, followed by IN.PACT and Lutonix (Ranger vs IN.PACT vs Lutonix: 1089 [745-2170] pmol/mg vs 638 [160-2075] pmol/mg vs 25 [10-304] pmol/mg, respectively; p<0.0001). In the histopathological evaluation, the angle of severe medial smooth muscle cell loss was largest for Ranger followed by IN.PACT and Lutonix (12.8 [8.0-20.4] degree vs 1.4 [1.2-5.2] degree vs 0.8 [0.5-2.5] degree, respectively), with significant differences for Ranger vs IN.PACT (p=0.007) and Ranger vs Lutonix (p=0.002). However, paclitaxel concentrations of distal skeletal muscles were lowest for Lutonix, followed by Ranger and IN.PACT (12 [1-58] pmol/mg vs 15 [13-21] pmol/mg vs 42 [19-108] pmol/mg, respectively, p<0.0001). The numbers of arteries with downstream DCB effects were highest for IN.PACT, followed by Ranger and Lutonix (Ranger vs IN.PACT vs Lutonix, 3 [3-4] vs 4 [3-7] vs 2 [1-2], respectively), which was consistent with the measured tissue paclitaxel concentrations. CONCLUSION: These findings suggest that Ranger demonstrates the strongest paclitaxel effect, as well as the second-best effect regarding distal particulate embolization, making it a good treatment option for patients with peripheral artery disease among the 3 DCBs evaluated in the current study. Further clinical head-to-head comparisons with larger numbers of patients are needed to explore which DCB is the most effective and safe treatment option.Clinical Impact:The findings of the current preclinical study suggests that Ranger demonstrates the strongest paclitaxel effect, as well as the second-best effect regarding distal particulate embolization making it a good treatment for patients with intermittent claudication and chronic limb-threatening ischemia.

Poor Below Knee Runoff Impacts Femoropopliteal Stent Failure and Fluoropolymer Antithrombotic Effect in Healthy Swine Model.

OBJECTIVE: Poor below knee (BTK) runoff is a predictor of stent failure after endovascular femoropopliteal artery treatment; however, lack of pathological evaluation has prevented characterisation of stent failure. The study aimed to investigate the impact of poor BTK runoff and the antithrombotic effect of the polymer of fluoropolymer coated paclitaxel eluting stents (FP-PESs) in a healthy swine femoropopliteal artery model. METHODS: FP-PESs and bare metal stents (BMSs) and FP-PES and polymer free paclitaxel coated stents (PF-PCSs) were implanted in the bilateral femoral arteries of healthy swine (n = 6, respectively) following coil embolisation in both tibial arteries to induce poor BTK runoff. Histological assessment and intravascular imaging device evaluation were performed at one month. The Japanese Association for Laboratory Animal Science approved the study protocol (reference number: IVT22-90). RESULTS: Optical coherence tomography showed significantly lower percent area stenosis in FP-PES compared with BMS (37.3%, [interquartile range (IQR), 25.6 - 54.3] % vs. 92.5% [IQR, 75.5 - 96.1] %, respectively, p = .031), and PF-PCS (8.3% [IQR, 4.5 - 27.0] % vs. 31.2% [IQR, 23.3 - 52.2] %, respectively, p = .031). Histopathological evaluation demonstrated that thin fibrin attachment without re-stenosis was the most dominant neointimal tissue characteristic in FP-PES. On the other hand, neointimal tissue characteristics with significant restenosis of BMS and PF-PCS were mainly organising or organised thrombus. CONCLUSION: Organising and or organised thrombus attachment due to poor BTK runoff was the main cause of in stent restenosis of the swine femoral artery. FP-PES demonstrated the least percent area stenosis, suggesting the importance of the antithrombotic effect of polymer.

Association Between Statin Use Prior to Admission and Lower Coronavirus Disease 2019 (COVID-19) Severity in Patients With Cardiovascular Disease or Risk Factors.

BACKGROUND: Cardiovascular diseases and/or risk factors (CVDRF) have been reported as risk factors for severe coronavirus disease 2019 (COVID-19).Methods and Results:In total, we selected 693 patients with CVDRF from the CLAVIS-COVID database of 1,518 cases in Japan. The mean age was 68 years (35% females). Statin use was reported by 31% patients at admission. Statin users exhibited lower incidence of extracorporeal membrane oxygenation (ECMO) insertion (1.4% vs. 4.6%, odds ratio [OR]: 0.295, P=0.037) and septic shock (1.4% vs. 6.5%, OR: 0.205, P=0.004) despite having more comorbidities such as diabetes mellitus. CONCLUSIONS: This study suggests the potential benefits of statins use against COVID-19.

The recovery time required for rat joint contractures treated with joint fixation with unweighting of the hind limbs.

[Purpose] The purpose of this study was to investigate whether the recovery period of joint contractures differ between treatments with joint fixation with unweighting of the hind limbs and joint fixation alone. [Subjects and Methods] Twelve 10-week-old male Wistar rats were divided into a group with joint fixation of the right ankle joints in full plantar flexion for 1 week (F) and a group with joint fixation and hind limb suspension (FS). Thereafter, the rats were removed from the joint fixation and hind limb suspension for 2 weeks. The ankle dorsiflexion angles during the recovery process of the joint contractures were measured. [Results] The ankle dorsiflexion angles of the F group from the day joint fixation was removed to the seventh recovery day were significantly lower than the pre-experiment values. The ankle dorsiflexion angles of the FS group from the day joint fixation and hind limb suspension were removed to the 10th recovery day were significantly lower than the pre-experiment values. [Conclusion] We found that the period required for the recovery of joint contractures treated with joint fixation with unweighting of the hind limbs was longer than that with joint fixation alone.

Study on change of rat soleus extensibility caused by reduction in joint movement with unweighting of the hind limbs.

[Purpose] The purpose of this study was to investigate the change in soleus extensibility from a joint contracture caused by a reduction in joint movement with unweighted lower limbs and to interpret the results to aid in the treatment of human joint contractures. [Subjects and Methods] The subjects of this study were twenty-one 10-weeks-old male Wistar rats. Twenty-one rats were divided into one control (C) and two experimental groups. The first experimental group included fixed right ankle joints in full plantar flexion (F). The second experimental group's hind limbs were suspended and the right ankle joints were fixed in full plantar flexion (FS). The period of this study was one week. On the first and last day of this study, all of the rats' ankle dorsiflexion angles were measured. On the last day of this study, all of the rats' soleus extensibilities were measured. [Results] On the last day of this study, the ankle dorsiflexion angles and the soleus extensibility in the FS group were significantly more decreased than those of the F group. [Conclusion] It was shown that the skeletal muscle extensibility from joint contracture caused by reduction of joint movement with unweighted lower limbs was more markedly decreased than that from joint contracture caused by reduction of joint movement.

Synthesis and Biodegradation of Poly(l-lactide-co-ß-propiolactone).

Although the copolymerizations of l-lactide (LA) with seven- or six-membered ring lactones have been extensively studied, the copolymerizations of LA with four-membered ring lactones have scarcely been reported. In this work, we studied the copolymerization of LA with ß-propiolactone (PL) and the properties of the obtained copolymers. The copolymerization of LA with PL was carried out using trifluoromethanesulfonic acid as a catalyst and methanol as an initiator to produce poly(LA-co-PL) with Mn of ~50,000 and PL-content of 6-67 mol %. The Tg values of the copolymers were rapidly lowered with increasing PL-contents. The Tm and ΔHm of the copolymers gradually decreased with increasing PL-contents, indicating their decreased crystallinity. Biodegradation test of the copolymers in compost demonstrated their improved biodegradability in comparison with the homopolymer of LA.

Effects of joint immobilization and hindlimb unloading on collagen fibers of soleus muscles in rats.

[Purpose] The purpose of this study was to determine whether collagen fibers cause a difference in a contracture resulting from a combination of joint fixation and hindlimb unloading as compared to joint fixation only. [Subjects and Methods] The subjects of this study were 21 female Wistar rats divided into 4 groups as follows: Control Group (CON, n=7); Joint Fixation Group (JF, n=7), Hindlimb Unloading Group (HU, n=7), Joint Fixation Plus Hindlimb Unloading Group (JF+HU, n=7). This study was conducted for 1-week. Ankle joint range of motion and positive areas of collagen using fluorescent stain were analyzed. [Results] Ankle joint range of motion in JF + HU showed an increase compared to that of JF. Positive areas of Type I collagen in JF+HU showed an increase as compared with that of JF. [Conclusion] The results of this study suggested that the difference in a contracture caused by only joint immobilization and by a combination of joint immobilization and hindlimb unloading were significantly associated with Type I collagen.

A study of the relationships of changes in pain and gait after tourniquet-induced ischemia-reperfusion in rats.

[Purpose] The purpose of this study was to determine the relationships of changes in pain and gait after ischemia reperfusion was induced by tourniquet in rats. [Subjects and Methods] The subjects were six ten-week-old male Wistar rats. Ischemia was induced in the left lower limbs of the experimental rats at a pressure of 300 mmHg for 90 minutes. Pain behavior evaluations were measured using the von Frey test in all the rats' hind limbs. A consistently increasing plantar stimulus was applied until the rats exhibited an escape behavior. For the evaluation of gait, a two-dimensional motion analysis system was used to measure the distance from the calcaneus to the floor (DCF) and toe extension angle (TEA) during gait. The evaluations were performed in the normal state, 3 hours after ischemia-reperfusion, and daily until 7 days after ischemia-reperfusion. [Results] Compared with the normal state, the means of the pain threshold showed a significant decrease until 4 days after ischemia. In addition, both TEA and DCF continued to show a significant decrease at 7 days after ischemia as compared with the normal state. [Conclusion] This study revealed that hyperalgesia occurs after ischemia-reperfusion, and recovery of hyperalgesia occurred earlier than gait dysfunction recovery.

Effects of skin stretching without joint movement on skin extensibility of rats.

[Purpose] This study examined the possibility of maintaining skin extensibility by stretching the skin involved in disuse joint contracture. [Subjects and Methods] The study was carried out using 18 male Wistar rats. The rats were randomly allocated to three groups. The control group received no intervention for the right ankle joint, the fixation group received one-week's fixation of the right ankle joint in maximum plantar-flexion with a cast, and the stretching group received continuous stretching of the skin over the Achilles tendon for 30 min once daily for one week with the cast removed during the skin stretching, but the joint was not moved. On the final day, skin extensibility of the skin from the posterior aspect of the ankle joint was determined using a tensile strength tester and a length-tension curve. [Results] Statistical analysis of the data revealed significant differences in the skin extensibility among the three groups. The stretching group showed significantly greater improvement of skin extensibility than the fixation group. [Conclusion] Skin stretching without moving the joint was demonstrated to be useful for maintaining skin extensibility.

Study on changes in skin extensibility during the development of joint contracture due to joint immobilization in rats.

[Purpose] The purpose of this study was to elucidate whether skin extensibility decreases when a contracture develops as a result of joint immobilization. [Subjects] This study was conducted on six female Wistar rats. [Methods] The rats were divided into two experimental groups. In the immobilized group, the right ankle joints were immobilized in complete plantar flexion by plaster casts for two weeks. In the control group, the left ankle joints had no intervention. On the final day, skin extensibility was determined from a length-tension curve by collecting skin from the posterior aspect of the ankle joint and using a tensile strength tester. [Results] Compared with the control group, the immobilized group showed a significant decrease in skin extensibility. [Conclusion] The results demonstrated that the extensibility of the skin itself decreases when joint contracture develops.

Effect of stretch on improvement of muscular contractures in rats.

[Purpose] The purpose of this study was to investigate how a stretching torque affects muscular contractures. [Subjects] The subjects of this study were 48 male Wistar rats. [Methods] Subjects were divided into 4 groups as follows: Group 1 was the control; Group 2 had muscles in continuous fixation; Group 3 had muscles stretched in the direction of dorsiflexion by a spring balancer set at a torque of 0.3N for a period of 30 minutes after continuous fixation; and Group 4 had muscles stretched in the direction of dorsiflexion by a spring balancer set at a torque of 3.0N for a period of 30 minutes after continuous fixation. Joint fixation periods were for 2 and 4-weeks. Ankle joint range of motion and soleus flexibility were analyzed. [Results] For the 2-week joint fixation, soleus flexibility in Group 4 showed an increase compared with that of Group 3. For both fixation periods, range of motion in Group 4 showed an increase compared with that of Group 3. [Conclusion] For both fixation periods, stretching improved joint range of motion. In the 2-week joint fixation, soleus flexibility improved. However, soleus flexibility did not improve in the 4-week joint fixation.

Effect of skin resection on the improvement of joint contractures in rats.

The effect of skin resection on joint contracture was determined by comparing the first measurement of range of motion after cast removal and the second measurement after the skin resection. This study aimed to verify that both the joint movement during the measurement and skin affect range of motion. [Subjects] Twelve female Wistar rats were used. [Methods] The right hind limb ankle of each rat was immobilized in complete plantar flexion in a cast. In the resection group (n = 6), the skin of the right hind limb ankle was removed surgically, but not in the non-resection group (n = 6). In the resection group, the first measurement of the dorsiflexion angle was obtained after the cast was removed, and the second measurement was obtained after skin resection. In the non-resection group, both measurements of the dorsiflexion angle were obtained soon after the cast was removed. [Results] Compared with the non-resection group, the resection group showed a significant increase between the first and second measurements of range of motion. [Conclusion] These results show that range of motion is substantially affected by skin, in addition to joint movement, during measurement.

Effect of an Intermittent Eight-hour Joint Fixation Period on Joint Contractures in Rats.

[Purpose] It has been shown that an eight-hour joint fixation daily (i.e. for the remaining 16 hours, rats were set free in the cage without joint fixation) for one week induces a joint contracture. The purpose of this study was to investigate the difference between an 8 hours continuous joint fixation and an 8 hours intermittent joint fixation per day (two 4 hours joint fixations) in the development of joint contractures in rats. [Subjects and Methods] The subjects of this study were 34 Wistar rats. On the first day, all of the rats' right ankle dorsiflexion angles were measured. The right hind limbs of all the rats were fixed in plantar flexion, and then the rats were divided into three groups as follows: Group 1 was in continuous fixation for 24 hours a day; Group 2 was in continuous fixation for 8 hours a day; Group 3 was in intermittent fixation for 8 hours a day. The fixation was performed daily for seven consecutive days. On the last day, ankle dorsiflexion angles of all the rats were measured after the casts had been removed. [Results] The development of joint contractures was statistically significant for Groups 1 and 2. [Conclusion] Our results indicate that 8 hours a day of continuous fixation induces a joint contracture, but 8 hours a day of intermittent fixation does not.

Preventive effects of antioxidants and exercise on muscle atrophy induced by ischemic reperfusion.

[Purpose] This study aimed to determine whether muscle atrophy induced by ischemic reperfusion injury in rats can be prevented by the administration of antioxidants and exercise. [Subjects] Rats were randomly divided into five groups: non-treated, ischemic, exercise, ascorbic acid and exercise, and tocopherol and exercise. [Methods] The relative weight ratio of the soleus muscle and the length of the soleus muscle fiber cross-section minor axis were used for the evaluation of muscle atrophy. Pain was assessed as the weight-bearing ratio of the ischemic side. A multiple comparison test and the paired t-test were used for the statistical analyses. [Results] Compared with the non-treated group, the relative weight ratios of the soleus muscle and the lengths of the soleus muscle fiber cross-section minor axis significantly decreased in the other groups. Excluding the non-treated group, the relative weight ratios of the soleus muscle were heaviest in the tocopherol and exercise group. Excluding the non-treated group, the lengths of the soleus muscle fiber cross-section minor axis were longest in the tocopherol and exercise group, followed by the ischemic, exercise, and ascorbic acid and exercise groups. The amount of antioxidant substances did not decrease on the weight-bearing ratio of the ischemic side. [Conclusion] In this study, using an experimental rat model, we confirmed that antioxidants and exercise effect muscle atrophy induced by ischemic reperfusion. The results show that muscle regeneration was facilitated by phagocytosis in the tocopherol and exercise group.

Biological differences of three paclitaxel- and sirolimus-coated balloons on coronary lesions in a rabbit model.

BACKGROUND: Drug-coated balloons (DCBs) are important treatment options for coronary artery disease; however, randomised controlled trials comparing various DCB technologies are sparse, and further investigations are needed. AIMS: This preclinical study aimed to histologically and biologically compare the drug effects and safety of a low-dose paclitaxel-coated DCB (PCB; AGENT), a regular-dose PCB (SeQuent Please NEO) and a sirolimus-coated DCB (SCB; MagicTouch). METHODS: The DCBs were inflated in the healthy iliac arteries of 18 rabbits, which were euthanised after 28 days. The treated iliac arteries and distal skeletal muscles were histopathologically evaluated, and drug concentrations were measured. RESULTS: In the histopathological evaluation, the medial smooth muscle cell loss score regarding depth, an indicator of drug efficacy, was significantly higher with AGENT and SeQuent Please NEO than with MagicTouch (4.0 [3.6-4.0] vs 3.7 [3.7-4.0] vs 2.2 [2.0-2.4]), with significant differences in comparisons between AGENT and MagicTouch (p<0.01) and between SeQuent Please NEO and MagicTouch (p<0.01). AGENT and SeQuent Please NEO showed comparable drug concentrations in the treated artery (p=0.61). In contrast, the drug concentrations in distal skeletal muscles were the highest for MagicTouch, followed by SeQuent Please NEO and AGENT (28.07 [13.19-52.46] ng/mg vs 0.66 [0.22-3.76] ng/mg vs 0.25 [0.04-3.23] ng/mg, respectively). CONCLUSIONS: This study demonstrated that PCBs might have higher efficacy and lower drug concentrations in distal skeletal muscles than the MagicTouch SCB. The efficacy of the AGENT low-dose PCB and the SeQuent Please NEO regular-dose PCB was comparable.

Histological evaluation of vascular changes after excimer laser angioplasty for neointimal formation after bare-metal stent implantation in rabbit iliac arteries.

BACKGROUND: Excimer laser is used to treat coronary artery disease, especially in case of lesions with thrombus and in-stent restenosis (ISR). However, there are no in vivo preclinical studies that have evaluated the pathological reactions of the vessel wall after excimer laser ablation. METHODS: Bare-metal stents were placed in the external iliac arteries of six healthy rabbits. Twenty-eight days later, excimer laser ablation was performed with low-power (45 (fluency)/25 (rate)) in one side, and high-power (60/40) in the opposite side, followed by optical coherence tomography (OCT) evaluation. Rabbits were sacrificed 15 min after the procedure, and histological assessment was performed. RESULTS: Morphometry analysis of OCT showed similar stent and lumen size between low-power and high-power group. Histological evaluation suggested endothelial cell loss, fibrin deposition, and tissue loss. The low-power group showed significantly less pathological changes compared with the high-power group: angle of endothelial cell loss, 32.4° vs. 191.7° (interquartile range, 8.8°-131.7° vs. 125.7°-279.5°; p < 0.01); fibrin deposition, 1.1° vs. 59.6° (0.0°-70.4° vs. 31.4°-178.4°; p = 0.03); and tissue loss 0.0° vs. 18.2° (0.0°-8.7° vs. 0.0°-42.7°; p = 0.03). CONCLUSIONS: The pathological changes in neointima were more prominent after high-power excimer laser ablation than after low-power excimer laser. To improve safety in clinical practice, understanding the pathological changes of tissues after excimer laser in lesions with ISR is essential.

A novel tdTomato transgenic mouse model to visualize FAP-positive cancer-associated fibroblasts.

Fibroblast activation protein (FAP) generally shows low or undetectable expression in most normal tissues but is highly expressed in fibroblasts in almost all carcinomas. FAP is one of the potential molecules to detect activated fibroblasts and has multiple roles in tumour progression. We generated transgenic mice that specifically expressed tdTomato along with FAP promoter activity. Coculturing a mouse gastric cancer cell line and FAP-tdTomato transgenic mouse-derived fibroblasts showed that tdTomato expression was elevated in the cocultured fibroblasts. Moreover, stomach wall transplanted tumours in mice also showed FAP-tdTomato expression in fibroblasts of the stomach and each metastatic legion. These results indicated that FAP-tdTomato expression in fibroblasts was elevated by stimulation through the interaction with cancer cells. Functionally, collagen production was increased in FAP/tdTomato-positive fibroblasts cocultured with mouse cancer cells. These FAP-tdTomato transgenic mice have the potential to be used to investigate real-time FAP dynamics and the importance of FAP expression in tumour development.

Pathological Analysis of Medial and Intimal Calcification in Lower Extremity Artery Disease: Impact of Hemodialysis.

Background: The prevalence and degree of lower extremity artery disease in hemodialysis (HD) patients is higher than in the general population. However, the pathological features have not yet been evaluated. Objectives: The aim of the study was: 1) to compare lesion characteristics of lower extremity artery disease in HD vs non-HD patients; and 2) to determine factors associated with severe medial calcification. Methods: Seventy-seven lower limb arteries were assessed from 36 patients (median age 77 years; 23 men; 21 HD and 15 non-HD) who underwent autopsy or lower limb amputation. Arteries were serially cut at 3- to 4-mm intervals creating 2,319 histological sections. Morphometric analysis and calcification measurements were performed using ZEN software. Calcification with a circumferential angle (arc) ≥180° was defined as severe calcification. Multivariable logistic regression was used to identify risk factors for severe medial calcification. Results: The degree of the medial calcification arc was significantly higher in the HD group compared to the non-HD group (P < 0.0001). In the multivariable analysis, HD was associated with severe medial calcification in below-the-knee lesions (OR: 17.1; P = 0.02). The degree of intimal calcification in above-the-knee lesions was also significantly higher in HD patients with a higher prevalence of advanced atherosclerotic plaque (P = 0.02). The prevalence of severe bone formation was more common in the HD patients (P = 0.01). Conclusions: Hemodialysis patients demonstrated a higher degree of medial and intimal calcification compared with non-HD patients. The difference was more prominent in the medial calcification of below-the-knee lesions.