Pathological Changes in the Gastrocnemius Muscle throughout Hind Limb Ischemia Modeling in Rats.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.

KANK4 Promotes Arteriogenesis by Potentiating VEGFR2 Signaling in a TALIN-1-Dependent Manner.

BACKGROUND: Arteriogenesis plays a critical role in maintaining adequate tissue blood supply and is related to a favorable prognosis in arterial occlusive diseases. Strategies aimed at promoting arteriogenesis have thus far not been successful because the factors involved in arteriogenesis remain incompletely understood. Previous studies suggest that evolutionarily conserved KANK4 (KN motif and ankyrin repeat domain-containing proteins 4) might involve in vertebrate vessel development. However, how the KANK4 regulates vessel function remains unknown. We aim to determine the role of endothelial cell-specifically expressed KANK4 in arteriogenesis. METHODS: The role of KANK4 in regulating arteriogenesis was evaluated using Kank4-/- and KANK4iECOE mice. Molecular mechanisms underlying KANK4-potentiated arteriogenesis were investigated by employing RNA transcriptomic profiling and mass spectrometry analysis. RESULTS: By analyzing Kank4-EGFP reporter mice, we showed that KANK4 was specifically expressed in endothelial cells. In particular, KANK4 displayed a dynamic expression pattern from being ubiquitously expressed in all endothelial cells of the developing vasculature to being explicitly expressed in the endothelial cells of arterioles and arteries in matured vessels. In vitro microfluidic chip-based vascular morphology analysis and in vivo hindlimb ischemia assays using Kank4-/- and KANK4iECOE mice demonstrated that deletion of KANK4 impaired collateral artery growth and the recovery of blood perfusion, whereas KANK4 overexpression leads to increased vessel caliber and blood perfusion. Bulk RNA sequencing and Co-immunoprecipitation/mass spectrometry (Co-IP/MS) analysis identified that KANK4 promoted EC proliferation and collateral artery remodeling through coupling VEGFR2 (vascular endothelial growth factor receptor 2) to TALIN-1, which augmented the activation of the VEGFR2 signaling cascade. CONCLUSIONS: This study reveals a novel role for KANK4 in arteriogenesis in response to ischemia. KANK4 links VEGFR2 to TALIN-1, resulting in enhanced VEGFR2 activation and increased EC proliferation, highlighting that KANK4 is a potential therapeutic target for promoting arteriogenesis for arterial occlusive diseases.

Downregulated calmodulin expression contributes to endothelial cell impairment in diabetes.

Endothelial dysfunction, a central hallmark of cardiovascular pathogenesis in diabetes mellitus, is characterized by impaired endothelial nitric oxide synthase (eNOS) and NO bioavailability. However, the underlying mechanisms remain unclear. Here in this study, we aimed to identify the role of calmodulin (CaM) in diabetic eNOS dysfunction. Human umbilical vein endothelial cells and murine endothelial progenitor cells (EPCs) treated with high glucose (HG) exhibited downregulated CaM mRNA/protein and vascular endothelial growth factor (VEGF) expression with impeded eNOS phosphorylation and cell migration/tube formation. These perturbations were reduplicated in CALM1-knockdown cells but prevented in CALM1-overexpressing cells. EPCs from type 2 diabetes animals behaved similarly to HG-treated normal EPCs, which could be rescued by CALM1-gene transduction. Consistently, diabetic animals displayed impaired eNOS phosphorylation, endothelium-dependent dilation, and CaM expression in the aorta, as well as deficient physical interaction of CaM and eNOS in the gastrocnemius. Local CALM1 gene delivery into a diabetic mouse ischemic hindlimb improved the blunted limb blood perfusion and gastrocnemius angiogenesis, and foot injuries. Diabetic patients showed insufficient foot microvascular autoregulation, eNOS phosphorylation, and NO production with downregulated CaM expression in the arterial endothelium, and abnormal CALM1 transcription in genome-wide sequencing analysis. Therefore, our findings demonstrated that downregulated CaM expression is responsible for endothelium dysfunction and angiogenesis impairment in diabetes, and provided a novel mechanism and target to protect against diabetic endothelial injury.

Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model.

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

Human and African ape myosin heavy chain content and the evolution of hominin skeletal muscle.

Humans are unique among terrestrial mammals in our manner of walking and running, reflecting 7 to 8 Ma of musculoskeletal evolution since diverging with the genus Pan. One component of this is a shift in our skeletal muscle biology towards a predominance of myosin heavy chain (MyHC) I isoforms (i.e. slow fibers) across our pelvis and lower limbs, which distinguishes us from chimpanzees. Here, new MyHC data from 35 pelvis and hind limb muscles of a Western gorilla (Gorilla gorilla) are presented. These data are combined with a similar chimpanzee dataset to assess the MyHC I content of humans in comparison to African apes (chimpanzees and gorillas) and other terrestrial mammals. The responsiveness of human skeletal muscle to behavioral interventions is also compared to the human-African ape differential. Humans are distinct from African apes and among a small group of terrestrial mammals whose pelvis and lower limb muscle is slow fiber dominant, on average. Behavioral interventions, including immobilization, bed rest, spaceflight and exercise, can induce modest decreases and increases in human MyHC I content (i.e. -9.3% to 2.3%, n = 2033 subjects), but these shifts are much smaller than the mean human-African ape differential (i.e. 31%). Taken together, these results indicate muscle fiber content is likely an evolvable trait under selection in the hominin lineage. As such, we highlight potential targets of selection in the genome (e.g. regions that regulate MyHC content) that may play an important role in hominin skeletal muscle evolution.

Chemerin regulates normal angiogenesis and hypoxia-driven neovascularization.

Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral factor inhibiting the vascularization of tumor grafts. We show here that overexpression of bioactive chemerin in mice results in a reduction of the density of the retinal vascular network during its development and in adults. Chemerin did not affect vascular sprouting during the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating the role of this receptor. Chemerin inhibited also neoangiogenesis in a model of pathological proliferative retinopathy, and in response to hind-limb ischemia. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the density of the retinal vasculature, suggesting the involvement of the PI3-kinase/AKT pathway in the chemerin-induced vessel regression process.

Hind limb muscles influence the architectural properties of long bones in frogs.

The Mechanostat Theory states that osteocytes sense both the intensity and directionality of the strains induced by mechanical usage and modulate the bone design accordingly. In long bones, this process may adapt anterior-posterior and lateral-medial strength to their mechanical environment showing regional specificity. Anuran species are ideal for analyzing the muscle-bone relationships related to the different mechanical stresses induced by their many locomotor modes and habitat uses. This work aimed to explore the relationships between indicators of the force of the most relevant muscles to locomotion and the mechanical properties of femur and tibia fibula in preserved samples of three anuran species with different habitat use (aquatic, arboreal) and locomotion modes (swimmer, jumper, walker/climber). For that purpose, we measured the anatomical cross-sectional area of each dissected muscle and correlated it with the moments of inertia and bone strength indices. Significant, species-specific covariations between muscle and bone parameters were observed. Pseudis platensis, the aquatic swimmer, showed the largest muscles, followed by Boana faber, the jumper and Phyllomedusa sauvagii, the walker/climber. As we expected, bigger muscles correlate with bone parameters in all the species. Nevertheless, smaller muscles also play an important role in bone design. In aquatic species, muscle interaction enhances mostly lateral bending strength throughout the femur and lateral and antero-posterior bending strength in the tibia fibula. In the jumper species, muscles affected the femur and tibia fibula mostly in anterior-posterior bending. In the walker/climber species, responses involving both antero-posterior and lateral bending strengths were observed in the femur and tibia fibula. These results show that bones will be more or less resistant to lateral and antero-posterior bending according to the different mechanical challenges of locomotion in aquatic vs. arboreal habitats. This study provides new evidence of the muscle-bone relationships in three frog species associated with their different locomotion and habitat uses, highlighting the crucial role of muscle in determining the architectural properties of bones.

Hind foot drumming: Volumetric micro-computed tomography investigation of the hind limb musculature of three African mole-rat species (Bathyergidae).

Several species of African mole-rats use seismic signalling by means of hind foot drumming for communication. The present study aimed to create three-dimensional reconstructions and compare volumetric measurements of 27 muscles of the hind limb of two drumming (Georychus capensis and Bathyergus suillus) and one non-drumming (Cryptomys hottentotus natalensis) species of African mole-rats. Diffusible iodine contrast-enhanced micro-computed tomography (diceCT) scans were performed on six specimens per species. Manual segmentation of the scans using VGMAX Studio imaging software allowed for individual muscles to be separated while automatically determining the volume of each muscle. The volume of the individual muscles was expressed as a percentage of the total hind limb volume and statistically compared between species. Subsequently, three-dimensional reconstructions of these muscles were created. Musculus gracilis anticus had a significantly larger percentage of the total hind limb muscle volume in both drumming species compared to the non-drumming C. h. natalensis. Furthermore, several hip and knee extensors, namely mm. gluteus superficialis, semimembranosus, gluteofemoralis, rectus femoris and vastus lateralis, had significantly larger muscle volume percentages in the two drumming species (G. capensis and B. suillus) compared to the non-drumming species. While not statistically significant, G. capensis had larger muscle volume percentages in several key hip and knee extensors compared to B. suillus. Additionally, G capensis had the largest summed percentage of the total hind limb volume in the hip flexor, hip extensor, knee extensor and ankle plantar flexor muscle groups in all the three species. This could be indicative of whole muscle hypertrophy in these muscles due to fast eccentric contractions that occur during hind foot drumming. However, significantly larger muscle volume percentages were observed in the scratch digging B. suillus compared to the other two chisel tooth digging species. Moreover, while not statistically significant, B. suillus had larger muscle volume percentages in several hip extensor and knee flexor muscles compared to G. capensis (except for m. vastus lateralis). These differences could be due to the large relative size of this species but could also be influenced by the scratch digging strategy employed by B. suillus. Therefore, while the action of hind foot drumming seems to influence certain key muscle volumes, digging strategy and body size may also play a role.

Gene therapy with Pellino-1 improves perfusion and decreases tissue loss in Flk-1 heterozygous mice but fails in MAPKAP Kinase-2 knockout murine hind limb ischemia model.

OBJECTIVES: In the United States, over 8.5 million people suffer from peripheral arterial disease (PAD). Previously we reported that Pellino-1(Peli1) gene therapy reduces ischemic damage in the myocardium and skin flaps in Flk-1 [Fetal Liver kinase receptor-1 (Flk-1)/ Vascular endothelial growth factor receptor-2/VEGFR2] heterozygous (Flk-1+/--) mice. The present study compares the angiogenic response and perfusion efficiency following hind limb ischemia (HLI) in, Flk-1+/- and, MAPKAPKINASE2 (MK2-/-) knockout (KO) mice to their control wild type (WT). We also demonstrated the use of Peli1 gene therapy to improve loss of function following HLI. STUDY DESIGN AND METHODS: Femoral artery ligation (HLI) was performed in both Flk-1+/- and MK2-/- mice along with their corresponding WT. Another set of Flk-1+/- and MK2-/- were injected with either Adeno-LacZ (Ad.LacZ) or Adeno-Peli1 (Ad.Peli1) after HLI. Hind limb perfusion was assessed by laser doppler imaging at specific time points. A standardized scoring scale is used to quantify the extent of ischemia. Histology analysis performed includes capillary density, fibrosis, pro-angiogenic and anti-apoptotic proteins. RESULTS: Flk-1+/- and MK2-/- had a slower recovery of perfusion efficiency in the ischemic limbs than controls. Both Flk-1+/- and MK2-/- KO mice showed decreased capillary density and capillary myocyte ratios with increased fibrosis than their corresponding wild types. Ad.Peli1 injected ischemic Flk-1+/- limb showed improved perfusion, increased capillary density, and pro-angiogenic molecules with reduced fibrosis compared to Ad.LacZ group. No significant improvement in perfusion was observed in MK2-/- ischemic limb after Ad. Peli1 injection. CONCLUSION: Deletion of Flk-1 and MK2 impairs neovascularization and perfusion following HLI. Treatment with Ad. Peli1 results in increased angiogenesis and improved perfusion in Flk-1+/- mice but fails to rectify perfusion in MK2 KO mice. Overall, Peli1 gene therapy is a promising candidate for the treatment of PAD.

Enhancing lymphangiogenesis and lymphatic drainage to vascularized lymph nodes with nanofibrillar collagen scaffolds.

BACKGROUND: This study investigated the effect of nanofibrillar collagen scaffold (BioBridge) implantation from the affected limb to the unaffected contralateral femoral vein or lymph node in a rat model. METHODS: Hind limb lymphedema in Lewis rats was created with lymphadenectomy and inguinal circumcision without radiation. The volumetric difference (greater than 5%) using computed tomography and indocyanine green fluorescence evaluated the progress of lymphedema at 4 weeks. The lymphedema rats have separated into Group I: Controls; Group II: implanted BioBridge to the contralateral femoral vein; and Group III: implanted BioBridge to the contralateral inguinal lymph node. RESULTS: A total of 14 of 30 (46.7%) rats developed hind limb lymphedema with a mean volume difference of 5.83 ± 0.99% and showed diffuse dermal backflow at 4 weeks postlymphadenectomy. Four weeks postimplantation of BioBridge, the mean volumetric difference was 5.62 ± 2.11%, 4.97 ± 0.59%, and -2.47 ± 2.37% in Group I, II, and III, respectively (p < 0.05). The dermal backflow on the affected limb increased in Groups I and II but decreased in Group III. CONCLUSIONS: Implantation of BioBridge from the affected limb to the contralateral inguinal lymph node significantly reduced the hind limb lymphedema at 4 weeks.

Complications and owner satisfaction associated with limb amputation in cats: 59 cases (2007-2017).

BACKGROUND: Limb amputation may be recommended in domestic cats following a severe injury or disease. The purpose of the study was to report the signalment, the complications, recovery outcome, owner satisfaction and expectations of domestic cats following limb amputation. RESULTS: Medical records of 3 specialty hospitals were reviewed for cats that received a single limb amputation in a 10 year period (2007-2017). These cat owners were contacted, and 59 owners completed surveys, comprising the study population. The most common reasons for limb amputation were neoplasia (54.2%, 32/59), traumatic injury (40.7%, 24/59), bone or joint infection (3.4%, 2/59), and thromboembolism (1.7%, 1/59). Thirty-four cats (57.6%) had postoperative complications. Of the fifty-nine surveys, 52.5% reported minor complications and 5.1% reported major complications. There were no differences in postoperative complication rates for thoracic versus pelvic limb amputations. All owners reported either excellent (77.9%, 46/59), good (20.3% 12/59), or fair (1.7%, 1/59) satisfaction with the procedure. Based on their previous experiences, 84.7% (50/59) of owners would elect limb amputation if medically warranted for another pet. The remaining 15.3% of owners who would not elect limb amputation again had experienced death of their pet with a median survival time of 183 days. CONCLUSION: Owners reported a positive satisfaction when considering complications, recovery outcome, and expectations. This study can be used by veterinarians to guide cat owners in the decision making process of limb amputation.

Thyroid Hormone Receptor α Controls the Hind Limb Metamorphosis by Regulating Cell Proliferation and Wnt Signaling Pathways in Xenopus tropicalis.

Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.

Myological reconstruction of the pelvic girdle and hind limb of the giant titanosaurian sauropod dinosaur Dreadnoughtus schrani.

Osteological correlates preserve more readily than their soft tissue counterparts in the fossil record; therefore, they can more often provide insight into the soft tissue anatomy of the organism. These insights can in turn elucidate the biology of these extinct organisms. In this study, we reconstruct the pelvic girdle and hind limb musculature of the giant titanosaurian sauropod Dreadnoughtus schrani based on observations of osteological correlates and Extant Phylogenetic Bracket comparisons. Recovered fossils of Dreadnoughtus exhibit remarkably well-preserved, well-developed, and extensive muscle scars. Furthermore, this taxon is significantly larger bodied than any titanosaurian for which a myological reconstruction has previously been performed, rendering this contribution highly informative for the group. All 20 of the muscles investigated in this study are sufficiently well supported to enable reconstruction of at least one division, including reconstruction of the M. ischiocaudalis for the first time in a sauropod dinosaur. In total, 34 osteological correlates were identified on the pelvic girdle and hind limb remains of Dreadnoughtus, allowing the reconstruction of 14 muscles on the basis of Level I or Level II inferences (i.e., not Level I' or Level II' inferences). Comparisons among titanosaurians suggest widespread myological variation, yet potential phylogenetic and other paleobiologic patterns are often obscured by fragmentary preservation, infrequent myological studies, and lack of consensus on the phylogenetic placement of many taxa. However, a ventrolateral accessory process is present on the preacetabular lobe of the ilium in all of the largest titanosauriforms that preserve this skeletal element, suggesting that the presence of this process (representing the origin of the M. puboischiofemoralis internus part II) may be associated with extreme body size. By identifying such myological patterns among titanosauriforms, we can begin to address specific evolutionary and biomechanical questions related to their skeletal anatomy, how they were capable of leaving wide-gauge trackways, and resulting locomotor attributes unique to this clade.

Fat grafting rescues radiation-induced joint contracture.

The aim of this study was to explore the therapeutic effects of fat grafting on radiation-induced hind limb contracture. Radiation therapy (RT) is used to palliate and/or cure a range of malignancies but causes inevitable and progressive fibrosis of surrounding soft tissue. Pathological fibrosis may lead to painful contractures which limit movement and negatively impact quality of life. Fat grafting is able to reduce and/or reverse radiation-induced soft tissue fibrosis. We explored whether fat grafting could improve extensibility in irradiated and contracted hind limbs of mice. Right hind limbs of female 60-day-old CD-1 nude mice were irradiated. Chronic skin fibrosis and limb contracture developed. After 4 weeks, irradiated hind limbs were then injected with (a) fat enriched with stromal vascular cells (SVCs), (b) fat only, (c) saline, or (d) nothing (n = 10/group). Limb extension was measured at baseline and every 2 weeks for 12 weeks. Hind limb skin then underwent histological analysis and biomechanical strength testing. Irradiation significantly reduced limb extension but was progressively rescued by fat grafting. Fat grafting also reduced skin stiffness and reversed the radiation-induced histological changes in the skin. The greatest benefits were found in mice injected with fat enriched with SVCs. Hind limb radiation induces contracture in our mouse model which can be improved with fat grafting. Enriching fat with SVCs enhances these beneficial effects. These results underscore an attractive approach to address challenging soft tissue fibrosis in patients following RT.

BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Canagliflozin impedes ischemic hind-limb recovery in the setting of diabetes.

There is a reported increased incidence of lower extremity amputations in individuals with diabetes who are treated with canagliflozin (an SGLT2 receptor inhibitor). It is unclear whether this is an unintended consequence of therapy, or whether canagliflozin can affect peripheral limb perfusion in the setting of underling arterial malperfusion. To evaluate this we explored the effect of canagliflozin on tissue recovery following unilateral hind-limb ischemia (HLI). Adult wildtype (+/+) and diabetic (db/db) mice were maintained on 8 weeks of a regular chow diet, or a chow diet containing canagliflozin (200 mg/kg). Following HLI, hind-limb appearance, function, and Doppler perfusion were serially evaluated. Gastrocnemius muscle fiber size and microvessel density were also evaluated 21 days following HLI. We observed that db/db that received a diet containing canagliflozin had significantly worse hind-limb function and appearance scores compared to both db/db mice that received a regular diet and +/+ mice that received a canagliflozin diet. At post-HLI day 21, db/db mice that received a canagliflozin diet also had decreased Doppler perfusion, gastrocnemius muscle fiber size, and microvessel density compared to +/+ mice that received a canagliflozin diet. These findings indicate that canagliflozin appears to impede ischemic peripheral tissue recovery and warrant further clinical investigation in individuals with diabetes and a history of peripheral artery disease.

Vascularized composite allotransplants as a mechanistic model for allograft rejection - an experimental study.

Vascularized composite allotransplants (VCAs) seem to have several unique features of clinical and experimental importance, including uniquely definable lymphatic drainage that can be easily accessed at the level of ipsilateral regional node beds. Thus, VCA offers a unique opportunity to assess the relative contribution of peripheral and secondary lymphoid tissue to the process of rejection. We transplanted hind limb grafts from C3H donors to six different groups of C57BL/6 recipients: Spleen+ Map3k14-/- ; Spleen- Map3k14-/- ; Spleen+ Node- Map3k14-/- ; and Spleen- Node- Map3k14-/- . As positive controls, we used Map3k14+/- with or without spleen. Map3k14+/- mice demonstrated an average graft survival of 9.6 and 9.2 days for Spleen- and Spleen+ Map3k14+/- , respectively. Rejection in the Map3k14-/- group was considerably delayed (28.4 days, P = 0.002) in all recipients. The Spleen- Map3k14-/- mice rejected their hind limb allografts in an even more delayed fashion compared to Spleen+ Map3k14-/- (54.4 days, P = 0.02). Histological analysis of skin showed that acute rejection in both Map3k14+/- mice groups was graded as Banff III or Banff IV. In the Map3k14-/- groups, rejection was graded as Banff III. We demonstrated that in the absence of lymph nodes, grafts reject in a delayed fashion. Also, splenectomy in alymphoplastic mice further extends graft survival, but does not eliminate rejection all together.

Chronic intermittent hypobaric hypoxia attenuates ischemic limb injury by promoting angiogenesis in mice.

This study aimed to evaluate the protective effect of chronic intermittent hypobaric hypoxia (CIHH) against limb ischemic injury. C57BL/6 mice were randomly divided into three groups: limb ischemic injury group (Ischemia, induced by ligation and excision of the left femoral artery), limb ischemia following CIHH pretreatment group (CIHH+Ischemia, simulated a 5000 m altitude hypoxia, 6 h per day for 28 days, before induction of hind-limb ischemia), and sham group (Sham). The blood flow in the mouse models of hind-limb ischemia was examined using laser doppler imaging. The functional and morphological performance of ischemic muscle was evaluated using contraction force and hematoxylin-eosin and Masson's trichrome staining. Angiogenesis was determined by immunohistochemistry staining of the endothelial markers CD31 and CD34. The protein expressions of angiogenesis-related genes were detected using Western blot assay. Chronic ischemia resulted in reduced blood perfusion, decreased contraction tension, and morphological destruction in gastrocnemius muscle. CIHH pretreatment increased the contractile force and muscle fiber diameter and decreased necrosis and fibrosis of the ischemic muscle. Also, CIHH significantly increased the density of CD31+ and CD34+ cells and promoted the expression of angiogenesis-related molecules in ischemic muscle. These data demonstrate that CIHH has a protective effect against chronic limb ischemia by promoting angiogenesis.

Altered temperature affect body condition and endochondral ossification in Bufo gargarizans tadpoles.

Bufo gargarizans is one kind of economic animals with higher medicinal value in China. In this study, B. gargarizans (Bufo gargarizans) tadpoles were reared at three different water temperature (15, 22 and 29 °C) from Gosner stages 28-46. We investigated the effects of temperature on growth, development, survival, metamorphic duration, size and skeletal ossification at Gosner stage 40, 42, and 46, as well as thyroid tissue reached metamorphic climax (Gs42). Besides, we examined the transcription levels of endochondral ossification-related genes in hind limb at metamorphic climax (Gs42). Our results showed that the growth and development of tadpoles conform to the temperature-size rule (TSR). While warm temperature resulted in the decrease in body size and hind limb length, and shorten larval period, cold temperature led to increase in body size and hind limb length but prolonged larval period. Histological examinations revealed that warm and cold temperatures caused damage to thyroid tissue. Also, warm and cold temperatures inhibited the degree of ossification with the double staining methodology. Additionally, the real-time PCR results suggested that warm and cold temperatures significantly up-regulated Runx2, VEGF and VEGFR mRNA levels, and down-regulated TRß, MMP9, MMP13 and Runx3 mRNA levels. The up-regulation of Dio2 level and down-regulation of Dio3 level were observed in warm temperature. TRα mRNA level was significantly increased in warm temperature, but decreased in cold temperature. Collectively, these observations demonstrated that warm and cold temperatures affected endochondral ossification in B. gargarizans tadpoles, which might influence their capacity to terrestrial locomotion.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.