Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Computational Model Exploring Characteristic Pattern Regulation in Periventricular Vessels.

The developing neocortical vasculature exhibits a distinctive pattern in each layer. In murine embryos, vessels in the cortical plate (CP) are vertically oriented, whereas those in the intermediate zone (IZ) and the subventricular zone (SVZ) form a honeycomb structure. The formation of tissue-specific vessels suggests that the behavior of endothelial cells is under a specific regulatory regime in each layer, although the mechanisms involved remain unknown. In the present study, we aimed to explore the conditions required to form these vessel patterns by conducting simulations using a computational model. We developed a novel model framework describing the collective migration of endothelial cells to represent the angiogenic process and performed a simulation using two-dimensional approximation. The attractive and repulsive guidance of tip cells was incorporated into the model based on the function and distribution of guidance molecules such as VEGF and Unc ligands. It is shown that an appropriate combination of guidance effects reproduces both the parallel straight pattern in the CP and meshwork patterns in the IZ/SVZ. Our model demonstrated how the guidance of the tip cell causes a variety of vessel patterns and predicted how tissue-specific vascular formation was regulated in the early development of neocortical vessels.

Essential Roles of Exocyst Complex Component 3-like 2 on Cardiovascular Development in Mice.

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

Effect of Downsized Screw Selection on Bone Healing and Postoperative Complications in Volar Plating of Distal Radius Fractures.

When performing volar plating of distal radius fractures, selecting downsized subchondral screws may prevent dorsal screw penetration (DSP), which is a risk factor for extensor tendon rupture. However, downsizing may cause loss of reduction or poor bone healing. This prospective study investigated the effect of downsized screw selection on bone healing and postoperative complications. A total of 115 patients with postoperative follow-up longer than 6 months comprised the study population. Using a depth gauge, screws that were 2-mm shorter than the measured value were selected. The DSP then was checked using dorsal tangential view (DTV) radiographs during surgery and at final follow-up. Baseline data included bone healing, loss of reduction of radiological parameters, DSP location, and postoperative complications. To assess DSP on DTV radiographs, the dorsal surface of the radius was divided into the radial and ulnar sides at the Lister tubercle, and each was further divided into 2 equal regions. These 4 regions were defined as zones 1 to 4 from the radial side. A total of 114 patients (99%) showed bone healing. Mean loss of reduction was approximately 1° and within 1 mm in radiological parameters. Eleven patients (9.6%) showed DSP during surgery or at final follow-up despite using 2-mm downsized screws. The most common site of DSP was zone 3. Extensor pollicis longus rupture occurred in 2 patients (1.7%) despite no DSP. Downsized screw selection provided a high rate of bone healing with minimum loss of reduction and a low complication rate. The extensor tendon can be torn regardless of DSP. [Orthopedics. 2021;44(2):e259-e265.].

Emerging Roles of PRDM Factors in Stem Cells and Neuronal System: Cofactor Dependent Regulation of PRDM3/16 and FOG1/2 (Novel PRDM Factors).

PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) (PR) homologous domain containing (PRDM) transcription factors are expressed in neuronal and stem cell systems, and they exert multiple functions in a spatiotemporal manner. Therefore, it is believed that PRDM factors cooperate with a number of protein partners to regulate a critical set of genes required for maintenance of stem cell self-renewal and differentiation through genetic and epigenetic mechanisms. In this review, we summarize recent findings about the expression of PRDM factors and function in stem cell and neuronal systems with a focus on cofactor-dependent regulation of PRDM3/16 and FOG1/2. We put special attention on summarizing the effects of the PRDM proteins interaction with chromatin modulators (NuRD complex and CtBPs) on the stem cell characteristic and neuronal differentiation. Although PRDM factors are known to possess intrinsic enzyme activity, our literature analysis suggests that cofactor-dependent regulation of PRDM3/16 and FOG1/2 is also one of the important mechanisms to orchestrate bidirectional target gene regulation. Therefore, determining stem cell and neuronal-specific cofactors will help better understanding of PRDM3/16 and FOG1/2-controlled stem cell maintenance and neuronal differentiation. Finally, we discuss the clinical aspect of these PRDM factors in different diseases including cancer. Overall, this review will help further sharpen our knowledge of the function of the PRDM3/16 and FOG1/2 with hopes to open new research fields related to these factors in stem cell biology and neuroscience.

Aging of the Vascular System and Neural Diseases.

Vertebrates have acquired complex high-order functions facilitated by the dispersion of vascular and neural networks to every corner of the body. Blood vessels deliver oxygen and nutrients to all cells and provide essential transport systems for removing waste products. For these functions, tissue vascularization must be spatiotemporally appropriate. Recent studies revealed that blood vessels create a tissue-specific niche, thus attracting attention as biologically active sites for tissue development. Each capillary network is critical for maintaining proper brain function because age-related and disease-related impairment of cognitive function is associated with the loss or diminishment of brain capillaries. This review article highlights how structural and functional alterations in the brain vessels may change with age and neurogenerative diseases. Capillaries are also responsible for filtering toxic byproducts, providing an appropriate vascular environment for neuronal function. Accumulation of amyloid ß is a key event in Alzheimer's disease pathogenesis. Recent studies have focused on associations reported between Alzheimer's disease and vascular aging. Furthermore, the glymphatic system and meningeal lymphatic systems contribute to a functional unit for clearance of amyloid ß from the brain from the central nervous system into the cervical lymph nodes. This review article will also focus on recent advances in stem cell therapies that aim at repopulation or regeneration of a degenerating vascular system for neural diseases.

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.

Interaction of the nervous system and vascular system is required for the proper assembly of the neocortex.

Mammalian neocortical development encompasses an entire set of events that leads to the generation of excitatory and inhibitory neurons from neural progenitors in the dorsal and ventral telencephalon, including cell proliferation, production of migratory precursors and their progeny, differentiation, and integration into circuits. During these processes, the developing neocortex acquires its vasculature by angiogenesis, a process consisting of proliferation of endothelial cells in existing blood vessels or vascular plexuses, and leading to formation of new blood vessels. Recent studies have suggested that neocortical angiogenesis progresses in a spatially and temporally restricted manner to construct a specialized vascular niche that supports ongoing neurogenesis during neocortical development. Here we review that periventricular blood vessels selectively influence neocortical progenitors behavior and neurogenesis, highlighting how CNS angiogenesis is utilized to construct neocortical cytoarchitecture.

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells.

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.

Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice.

Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.

Prdm16 is crucial for progression of the multipolar phase during neural differentiation of the developing neocortex.

The precise control of neuronal migration and morphological changes during differentiation is essential for neocortical development. We hypothesized that the transition of progenitors through progressive stages of differentiation involves dynamic changes in levels of mitochondrial reactive oxygen species (mtROS), depending on cell requirements. We found that progenitors had higher levels of mtROS, but that these levels were significantly decreased with differentiation. The Prdm16 gene was identified as a candidate modulator of mtROS using microarray analysis, and was specifically expressed by progenitors in the ventricular zone. However, Prdm16 expression declined during the transition into NeuroD1-positive multipolar cells. Subsequently, repression of Prdm16 expression by NeuroD1 on the periphery of ventricular zone was crucial for appropriate progression of the multipolar phase and was required for normal cellular development. Furthermore, time-lapse imaging experiments revealed abnormal migration and morphological changes in Prdm16-overexpressing and -knockdown cells. Reporter assays and mtROS determinations demonstrated that PGC1α is a major downstream effector of Prdm16 and NeuroD1, and is required for regulation of the multipolar phase and characteristic modes of migration. Taken together, these data suggest that Prdm16 plays an important role in dynamic cellular redox changes in developing neocortex during neural differentiation.

Isolated glenohumeral range of motion, excluding side-to-side difference in humeral retroversion, in asymptomatic high-school baseball players.

PURPOSE: Glenohumeral range of motion is correlated with shoulder capsular condition and is thus considered to be predictive of shoulder pathology. However, in throwing athletes, a side-to-side difference in humeral retroversion makes it difficult to evaluate capsular condition on the basis of glenohumeral range of motion measured by using the conventional technique. The purpose of this study was to measure isolated glenohumeral rotation, excluding side-to-side differences in humeral retroversion, in asymptomatic high-school baseball players. METHODS: A total of 195 high-school baseball players (52 pitchers and 143 position players; median age, 16 years) and 20 high-school non-throwing athletes (median age, 16 years) without any shoulder symptoms were enroled in this study. Glenohumeral external and internal rotations were measured by using both a conventional technique and our ultrasound-assisted technique. This technique, neutral rotation, was standardized on the basis of the ultrasonographically visualized location of the bicipital groove to exclude side-to-side differences in humeral retroversion from the calculated rotation angle. Intra- and inter-observer agreements of rotational measurements were evaluated by using intra-class correlation coefficients (ICCs). RESULTS: Isolated glenohumeral rotation measurements, excluding side-to-side differences in humeral retroversion, demonstrated excellent intra-observer (ICC > 0.89) and inter-observer (ICC > 0.78) agreements. Isolated glenohumeral internal rotation was significantly less in the dominant shoulder than in the non-dominant shoulder in asymptomatic baseball players (P < 0.001). Isolated glenohumeral external rotation in baseball players was significantly greater than in non-throwing athletes (P < 0.05). In the baseball players, humeral torsion in the dominant shoulder was significantly greater than that in the non-dominant shoulder (P < 0.001), indicating that the retroversion angle was greater in dominant shoulders than in non-dominant shoulders. CONCLUSIONS: Isolated glenohumeral external and internal rotations can be measured with high intra- and inter-observer reliability with the exclusion of side-to-side differences in humeral retroversion. Capsular and muscular changes in the throwing shoulder may be better evaluated by using our ultrasound-assisted technique. LEVEL OF EVIDENCE: Cross-sectional study, Level III.

Deletion of Prdm8 impairs development of upper-layer neocortical neurons.

Upper-layer (UL) neocortical neurons are the most prominent distinguishing features of the mammalian neocortex compared with those of the avian dorsal cortex and are vastly expanded in primates. However, little is known about the identities of the genes that control the specification of UL neurons. Here, we found that Prdm8, a member of the PR (PRDI-BF1 and RIZ homology) domain protein family, was specifically expressed in the postnatal UL neocortex, particular those in late-born RORß-positive layer IV neurons. We generated homozygous Prdm8 knockout (Prdm8 KO) mice and found that the deletion of Prdm8 causes growth retardation and a reduced brain weight, although the brain weight-to-body weight ratio is unchanged at postnatal day 8 (P8). Immunohistochemistry showed that the relative UL thickness, but not the thickness of the deep layer (DL), was significantly reduced in Prdm8 KO mice compared with wild-type (WT) mice. In addition, we found that a number of late-born Brn2-positive UL neurons were significantly decreased in Prdm8 KO mice. To identify genes regulated by Prdm8 during neocortical development, we compared expression profiling analysis in Prdm8 KO and WT mice, and identified some candidate genes. These results suggest that the proper expression of Prdm8 is required for the normal development and construction of UL neurons in the mammalian neocortex.

Clinical results of arthroscopic superior capsule reconstruction for irreparable rotator cuff tears.

PURPOSE: The objective of this study was to investigate the clinical outcome and radiographic findings after arthroscopic superior capsule reconstruction (ASCR) for symptomatic irreparable rotator cuff tears. METHODS: From 2007 to 2009, 24 shoulders in 23 consecutive patients (mean, 65.1 years) with irreparable rotator cuff tears (11 large, 13 massive) underwent ASCR using fascia lata. We used suture anchors to attach the graft medially to the glenoid superior tubercle and laterally to the greater tuberosity. We added side-to-side sutures between the graft and infraspinatus tendon and between the graft and residual anterior supraspinatus/subscapularis tendon to improve force coupling. Physical examination, radiography, and magnetic resonance imaging (MRI) were performed before surgery; at 3, 6, and 12 months after surgery; and yearly thereafter. Average follow-up was 34.1 months (24 to 51 months) after surgery. RESULTS: Mean active elevation increased significantly from 84° to 148° (P < .001) and external rotation increased from 26° to 40° (P < .01). Acromiohumeral distance (AHD) increased from 4.6 ± 2.2 mm preoperatively to 8.7 ± 2.6 mm postoperatively (P < .0001). There were no cases of progression of osteoarthritis or rotator cuff muscle atrophy. Twenty patients (83.3%) had no graft tear or tendon retear during follow-up (24 to 51 months). The American Shoulder and Elbow Surgeons (ASES) score improved from 23.5 to 92.9 points (P < .0001). CONCLUSIONS: ASCR restored superior glenohumeral stability and function of the shoulder joint with irreparable rotator cuff tears. Our results suggest that this reconstruction technique is a reliable and useful alternative treatment for irreparable rotator cuff tears. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

A new manual method for assessing elbow valgus laxity.

BACKGROUND: A screening of ulnar collateral ligament insufficiency is required for overhead throwers, since secondary pathologic changes result from an increased elbow valgus laxity. We developed a new manual method for assessing elbow valgus laxity and investigated the reliability of this method and its correlation with ultrasonographic assessment. METHODS: We defined elbow valgus laxity as the difference between the shoulder external rotation angle (ER angle) measured with the elbow in 90 degrees flexion and that measured with the elbow in extension because ER angle measured with the elbow in 90 degrees flexion includes elbow valgus laxity and ER angle with the elbow in extension does not include it. ER angle measurement with the elbow in extension involved the use of a custom arm holder. Three examiners each measured elbow valgus laxity by the new method in 5 healthy volunteers. Intraobserver and interobserver reliability was evaluated by calculating the intraclass correlation coefficient. We then assessed 19 high-school baseball players with no complaints of shoulder or elbow pain. Elbow ultrasonography was performed with a 10-MHz linear transducer with the elbow in 90 degrees flexion, and the forearm in the neutral position, and the width of the medial joint space at the level of the anterior bundle was measured. Elbow valgus laxity assessed by ultrasonography was defined as the difference between the medial joint space width with gravity stress and that without gravity stress. Increased elbow valgus laxity assessed by both our method and ultrasonography was defined as the difference between the laxity of the elbow on the throwing side and that on the contralateral side. Pearson's correlation coefficient (r) was calculated to evaluate the relationship between increased elbow valgus laxity obtained by our manual method and that by ultrasonography. RESULTS: Intraobserver reliability ranged from 0.92 to 0.98, and interobserver reliability was 0.70. The increased elbow valgus laxity assessed by our method was significantly correlated with that assessed by ultrasonographic assessment (P = 0.019, r = 0.53). CONCLUSIONS: Elbow valgus laxity can be assessed by our method. This method may be useful for screening for insufficiency of the ulnar collateral ligament.

Functional and structural outcomes of single-row versus double-row versus combined double-row and suture-bridge repair for rotator cuff tears.

BACKGROUND: Although previous biomechanical research has demonstrated the superiority of the suture-bridge rotator cuff repair over double-row repair from a mechanical point of view, no articles have described the structural and functional outcomes of this type of procedure. HYPOTHESIS: The structural and functional outcomes after arthroscopic rotator cuff repair may be different between the single-row, double-row, and combined double-row and suture-bridge (compression double-row) techniques. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: There were 206 shoulders in 201 patients with full-thickness rotator cuff tears that underwent arthroscopic rotator cuff repair. Eleven patients were lost to follow-up. Sixty-five shoulders were repaired using the single-row, 23 shoulders using the double-row, and 107 shoulders using the compression double-row techniques. Clinical outcomes were evaluated at an average of 38.5 months (range, 24-74 months) after rotator cuff repair. Postoperative cuff integrity was determined using Sugaya's classification of magnetic resonance imaging (MRI). RESULTS: The retear rates after arthroscopic rotator cuff repair were 10.8%, 26.1%, and 4.7%, respectively, for the single-row, double-row, and compression double-row techniques. In the subcategory of large and massive rotator cuff tears, the retear rate in the compression double-row group (3 of 40 shoulders, 7.5%) was significantly less than those in the single-row group (5 of 8 shoulders, 62.5%, P < .001) and the double-row group (5 of 12 shoulders, 41.7%, P < .01). Postoperative clinical outcomes in patients with a retear were significantly lower than those in patients without a retear for all 3 techniques. CONCLUSION: The additional suture bridges decreased the retear rate for large and massive tears. The combination of the double-row and suture-bridge techniques, which had the lowest rate of postoperative retear, is an effective option for arthroscopic repair of the rotator cuff tendons because the postoperative functional outcome in patients with a retear is inferior to that without retear.

Effect of shoulder abduction angle on biomechanical properties of the repaired rotator cuff tendons with 3 types of double-row technique.

BACKGROUND: After rotator cuff repair, the shoulder is immobilized in various abduction positions. However, there is no consensus on the proper abduction angle. PURPOSE: To assess the effect of shoulder abduction angle on the biomechanical properties of the repaired rotator cuff tendons among 3 types of double-row techniques. STUDY DESIGN: Controlled laboratory study. METHODS: Thirty-two fresh-frozen porcine shoulders were used. A simulated rotator cuff tear was repaired by 1 of 3 double-row techniques: conventional double-row repair, transosseous-equivalent repair, and a combination of conventional double-row and bridging sutures (compression double-row repair). Each specimen underwent cyclic testing followed by tensile testing to failure at a simulated shoulder abduction angle of 0° or 40° on a material testing machine. Gap formation and failure loads were measured. RESULTS: Gap formation in conventional double-row repair at 0° (1.2 ± 0.5 mm) was significantly greater than that at 40° (0.5 ± 0.3mm, P = .01). The yield and ultimate failure loads for conventional double-row repair at 40° were significantly larger than those at 0° (P < .01), whereas those for transosseous-equivalent repair (P < .01) and compression double-row repair (P < .0001) at 0° were significantly larger than those at 40°. The failure load for compression double-row repair was the greatest among the 3 double-row techniques at both 0° and 40° of abduction. CONCLUSION: Bridging sutures have a greater effect on the biomechanical properties of the repaired rotator cuff tendon at a low abduction angle, and the conventional double-row technique has a greater effect at a high abduction angle. CLINICAL RELEVANCE: Proper abduction position after rotator cuff repair differs between conventional double-row repair and transosseous-equivalent repair. The authors recommend the use of the combined technique of conventional double-row and bridging sutures to obtain better biomechanical properties at both low and high abduction angles.