The proper functioning of lymphatic valves is critical for unidirectional lymph transport. Valve development and maintenance depends on multiple genes in lymphatic endothelium, including those controlling the expression of 4 connexin (Cx) isoforms-Cx37, Cx47, Cx43, and Cx45. The relative importance of these isoforms for valve function is undefined, but primary human lymphedema is linked to loss-of-function mutations in Cx47 or Cx43, while deficiencies in Cx43 or Cx45 produce functional valve defects in mice. Tests of back leak and closure for single lymphatic valves from mice with selective deficiency of each Cx isoform revealed defects associated with the loss of Cx37 or Cx43, but not Cx47. Combined deletion of multiple isoforms, including Cx45 but not Cx47, produced even more severe valve defects in certain genotypes, sometimes with nearly complete regression of valves within 6 d. Back leak across connexin-deficient LVs correlated highly with gaps between the commissures formed by leaflet insertion into the vessel wall, indicating that connexin function may be critical for the formation and/or maintenance of leaflet commissures. Our results reveal the following hierarchy of Cx importance in valve function: Cx37 = Cx43 > Cx45 > Cx47 and predict that patients with loss of function mutations in Cx37 (GJA4) should develop lymphedema. We propose a general classification scheme describing 4 stages of progressive valve dysfunction.

Histotripsy is a focused ultrasound ablation therapy being developed for the treatment of liver tumors. A recent study investigating the feasibility of using histotripsy for the ablation of cholangiocarcinoma (CC), bile duct cancer that is difficult to treat with current therapies because of its location near critical structures and fibrous tissue, reported the feasibility of treating CC in an acute mouse model. Here, we investigate histotripsy for the in vivo ablation of CC in a chronic study using a 1-MHz transducer at an applied dose of 500 pulses/point. A pilot study determined that treating the CC tumors plus a 1- to 2-mm margin induced significant injuries to intestinal tissues, thus precluding the use of this strategy. Next, histotripsy was applied to CCs (n = 6) with the treatment contained to the tumor. Post-treatment, the ablation was visualized using ultrasound, and subjects were monitored over time. Histotripsy achieved an average of 73% reduction of tumor diameter 26 d after treatment, with no significant adverse events. Notably, three of six treated tumors were undetectable after 2.5 wk. The treated animals were found to have significantly increased tumor progression-free and overall survival. Overall, results indicate that histotripsy can be used as a safe and effective method for treating CC.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

With an overall 5-year survival rate of less than 9%, pancreatic cancer is a substantial cause of cancer related death in the United States. This high mortality has been associated with the immunosuppressive tumor microenvironment (TME) and the shortage of effective late-stage therapeutic options. Histotripsy is a non-thermal, non-ionizing, non-invasive cancer ablation modality that rapidly lyses targeted cells. To investigate how histotripsy can change the pancreatic TME, a Pan02 cell culture model of pancreatic adenocarcinoma was employed to determine the neoantigen release following histotripsy treatment and other standard ablation modalities. Histotripsy was found to release potential neoantigens at a level similar to other non-thermal ablations and superior to thermal ablation, which paralleled the increased innate immune system activation in vivo. For in vivo studies, subcutaneous Pan02 tumors in mice were treated with histotripsy. To monitor changes in the TME after treatment, rtPCR and flow cytometry were used and compared to untreated animals. Progression-free and overall survival were significantly improved with histotripsy treatment. Within 24 hours of treatment there was increased activation of the innate immune system and by 14 days there were decreased tumor-associated immune cell populations. This work illustrates the feasibility of using histotripsy to treat pancreatic cancer and shift the TME as well as providing mechanistic understanding into the activation of the innate immune system post-treatment. Future studies are needed to establish the mechanism of immunomodulation of the TME and the extent of potential systemic effects.

Connexin proteins form gap junctions controlling exchange of ions and small molecules between cells and play an important role in movement of lymph within lymphatic vessels. Connexin47 (CX47) is highly expressed in lymphatic endothelial cells and CX47 missense mutations, i.e., R260C, cosegregate with primary lymphedema in humans. However, studies utilizing CX47 knockout mice have failed to demonstrate any lymphatic anomalies. To unravel the lymphatic consequences of expressing a mutant CX47 protein, we used CRISPR technology to create a mouse carrying a Cx47 missense mutation (Cx47R259C) equivalent to the human CX47R260C missense mutation associated with human primary lymphedema. Intradermal Evans Blue dye injection identified a 2-fold increase in regional lymph nodes in homozygous Cx47R259C mice compared to wildtype, particularly in the jugular region (4.8 ± 0.4 and 2.0 ± 0.0, respectively, p<0.01). Associated lymphatic channels were increased in Cx47R259C mice and mesenteric lymph reflux occurred in homozygous Cx47R259C mice but not in wildtype. Contractility of superficial cervical lymphatics, assessed by pressure myography, was reduced in homozygous Cx47R259C mice compared to wildtype. In conclusion, our data are the first to demonstrate a role for the Cx47 protein in lymphatic anatomy and function. This phenotype is similar to that found with other valve deficient mouse mutants, e.g., in Foxc2. Of significance, this study is the first to use CRISPR technology to develop a pre-clinical model of primary lymphedema and demonstrates the importance of distinguishing between lack of and presence of mutant protein when developing clinically relevant animal models for translation of pre-clinical findings.

Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-free and general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

Key points Lymphatic valve defects are one of the major causes of lymph transport dysfunction; however, there are no accessible methods for quantitatively assessing valve function. This report describes a novel technique for quantifying lymphatic valve back‐leak. Postnatal endothelial‐specific deletion of connexin 43 (Cx43) in connexin 37 null (Cx37−/−) mice results in rapid regression of valve leaflets and severe valve dysfunction. This method can also be used for assessing the function of venous and lymphatic valves from various species, including humans. AbstractThe lymphatic system relies on robust, spontaneous contractions of collecting lymphatic vessels and one‐way secondary lymphatic valves to efficiently move lymph forward. Secondary valves prevent reflux and allow for the generation of propulsive pressure during each contraction cycle. Lymphatic valve defects are one of the major causes of lymph transport dysfunction. Genetic mutations in multiple genes have been associated with the development of primary lymphoedema in humans; and many of the same mutations in mice result in valve defects that subsequently lead to chylous ascites or chylothorax. At present the only experimental technique for the quantitative assessment of lymphatic valve function utilizes the servo‐null micropressure system, which is highly accurate and precise, but relatively inaccessible and difficult to use. We developed a novel, simplified alternative method for quantifying valve function and determining the degree of pressure back‐leak through an intact valve in pressurized, single‐valve segments of isolated lymphatic vessels. With this diameter‐based method, the competence of each lymphatic valve is challenged over a physiological range of pressures (e.g. 0.5–10cmH2O) and pressure back‐leak is extrapolated from calibrated, pressure‐driven changes in diameter upstream from the valve. Using mesenteric lymphatic vessels from C57BL/6J, Ub‐CreERT2;Rasa1fx/fx, Foxc2Cre/+, Lyve1‐Cre;Cx43fx/fx, and Prox1‐CreERT2;Cx43fx/fx;Cx37−/− mice, we tested our method on lymphatic valves displaying a wide range of dysfunction, from fully competent to completely incompetent. Our results were validated by simultaneous direct measurement of pressure back‐leak using a servo‐null micropressure system. Our diameter‐based technique can be used to quantify valve function in isolated lymphatic valves from a variety of species. This method also revealed that haplodeficiency in Foxc2 (Foxc2Cre/+) is not sufficient to cause significant valve dysfunction; however, postnatal endothelial‐specific deletion of Cx43 in Cx37−/− mice results in rapid regression of valve leaflets and severe valve dysfunction.

Rationale: Mutations in GJC2 and GJA1, encoding Cxs (connexins) 47 and 43, respectively, are linked to lymphedema, but the underlying mechanisms are unknown. Because efficient lymph transport relies on the coordinated contractions of lymphatic muscle cells (LMCs) and their electrical coupling through Cxs, Cx-related lymphedema is proposed to result from dyssynchronous contractions of lymphatic vessels. Objective: To determine which Cx isoforms in LMCs and lymphatic endothelial cells are required for the entrainment of lymphatic contraction waves and efficient lymph transport. Methods and Results: We developed novel methods to quantify the spatiotemporal entrainment of lymphatic contraction waves and used optogenetic techniques to analyze calcium signaling within and between the LMC and the lymphatic endothelial cell layers. Genetic deletion of the major lymphatic endothelial cell Cxs (Cx43, Cx47, or Cx37) revealed that none were necessary for the synchronization of the global calcium events that triggered propagating contraction waves. We identified Cx45 in human and mouse LMCs as the critical Cx mediating the conduction of pacemaking signals and entrained contractions. Smooth muscle–specific Cx45 deficiency resulted in 10- to 18-fold reduction in conduction speed, partial-to-severe loss of contractile coordination, and impaired lymph pump function ex vivo and in vivo. Cx45 deficiency resulted in profound inhibition of lymph transport in vivo, but only under an imposed gravitational load. Conclusions: Our results (1) identify Cx45 as the Cx isoform mediating the entrainment of the contraction waves in LMCs; (2) show that major endothelial Cxs are dispensable for the entrainment of contractions; (3) reveal a lack of coupling between lymphatic endothelial cells and LMCs, in contrast to arterioles; (4) point to lymphatic valve defects, rather than contraction dyssynchrony, as the mechanism underlying GJC2- or GJA1-related lymphedema; and (5) show that a gravitational load exacerbates lymphatic contractile defects in the intact mouse hindlimb, which is likely critical for the development of lymphedema in the adult mouse.

Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43-/- mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43∆LEC) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.

Venous valves (VVs) are critical for unidirectional blood flow from superficial and deep veins towards the heart. Congenital valve aplasia or agenesis may, in some cases, be a direct cause of vascular disease, motivating an understanding of the molecular mechanisms underlying the development and maintenance of VVs. Three gap junction proteins (Connexins), Cx37, Cx43, and Cx47, are specifically expressed at VVs in a highly polarized fashion. VVs are absent from adult mice lacking Cx37; however it is not known if Cx37 is required for the initial formation of valves. In addition, the requirement of Cx43 and Cx47 for VV development has not been studied. Here, we provide a detailed description of Cx37, Cx43, and Cx47 expression during mouse vein development and show by gene knockout that each Cx is necessary for normal valve development. The valve phenotypes in the knockout lines exhibit Cx-specific differences, however, including whether peripheral or central VVs are affected by gene inactivation. In addition, we show that a Cx47 null mutation impairs peripheral VV development but does not affect lymphatic valve formation, a finding of significance for understanding how some CX47 mutations cause inherited lymphedema in humans. Finally, we demonstrate a striking segregation of Foxc2 and NFATc1 transcription factor expression between the downstream and upstream faces, respectively, of developing VV leaflets and show that this segregation is closely associated with the highly polarized expression of Cx37, Cx43, and Cx47. The partition of Foxc2 and NFATc1 expression at VV leaflets makes it unlikely that these factors directly cooperate during the leaflet elongation stage of VV development.

Connexins (Cx), proteins that mediate direct intercellular communication, are essential for normal vascular development and function. Mutations in Cx47 have been reported to underlie cases of lymphedema in humans, however the cellular and molecular mechanisms responsible for dysfunction have been largely unexplored. This study examines an orthologous mutation in mouse Cx47 to one of these lymphedema‐causing mutations, an arginine to cysteine substitution (R259C) in a highly‐conserved region of the second extracellular loop. We transfected connexin‐deficient rat insulinoma (Rin) cells with wild‐type (WT) Cx47 or Cx47R259C either alone or in combination with WT Cx43. Evaluation of cell growth was performed using light microscopy, protein expression through Western blot and immunocytochemistry, and electrical coupling via dual whole‐cell voltage clamp. Expression of Cx47 in Rin cells resulted in plexiform cell growth. Cx47R259C and Cx43/Cx47R259C coexpressing Rin cells also displayed plexiform growth patterns, but exhibited more elongated gaps between cell groups. Surprisingly, in Cx43/Cx47R259C cells a multiple banding pattern for Cx43 was detected via Western blot and full length Cx43 was conspicuously absent. Immunocytochemistry of Cx43/Cx47R259C cells revealed a diffuse cytoplasmic staining pattern for Cx43 and an absence of readily identifiable gap junction plaques. Based on initial electrophysiological recordings, Cx47‐expressing Rin cells have a low incidence of electrical coupling. Together, these preliminary data suggest that Cx47 influences cell growth, and subtle alterations in pattern occur with mutation of Cx47 at R259C. Furthermore, Cx47R259C affects cellular processing of Cx43, which may be a potential molecular mechanism by which this orthologous mutation leads to lymphatic defects in humans. Support or Funding Information This work was supported by the following grants: National Institutes of Health (R21HL122443) and Heart, Lung, and Blood Training Grant (T32HL007249)

Connexins (Cxs), proteins that are vital for intercellular communication in vertebrates, have recently been shown to play a critical role in lymphatic development. However, our knowledge is currently limited regarding the functional relationships of Cxs with other proteins and signaling pathways. Cell culture studies have shown that Cx37 is necessary for coordinated activation of the transcription factor NFATc1, which cooperates with Foxc2 (another transcription factor) during lymphatic endothelial development. These data suggest that Cxs, Foxc2, and NFATc1 are part of a common developmental pathway. Here, we present a detailed characterization of Foxc2(+/-)Cx37(-/-) mice, demonstrating that lymphatic network architecture and valve formation rely on the concurrent embryonic expression and function of Foxc2 and Cx37. Foxc2(+/-)Cx37(-/-) mice have lymphedema in utero, exhibit craniofacial abnormalities, show severe dilation of intestinal lymphatics, display abnormal lacteal development, lack lymphatic valves, and typically die perinatally (outcomes not seen in Foxc2(+/-) or Cx37(-/-) mice separately). We provide a rigorous, quantitative documentation of lymphatic vascular network changes that highlight the specific structural alterations that occur in Foxc2(+/-)Cx37(-/-) mice. These data provide further evidence suggesting that Foxc2 and Cx37 are elements in a common molecular pathway directing lymphangiogenesis.

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.

BACKGROUND:Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS:We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS:Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

Venous valves play a crucial role in blood circulation, promoting the one-way movement of blood from superficial and deep veins towards the heart. By preventing retrograde flow, venous valves spare capillaries and venules from being subjected to damaging elevations in pressure, especially during skeletal muscle contraction. Pathologically, valvular incompetence or absence of valves are common features of venous disorders such as chronic venous insufficiency and varicose veins. The underlying causes of these conditions are not well understood, but congenital venous valve aplasia or agenesis may play a role in some cases. Despite progress in the study of cardiac and lymphatic valve morphogenesis, the molecular mechanisms controlling the development and maintenance of venous valves remain poorly understood. Here, we show that in valved veins of the mouse, three gap junction proteins (Connexins, Cxs), Cx37, Cx43, and Cx47, are expressed exclusively in the valves in a highly polarized fashion, with Cx43 on the upstream side of the valve leaflet and Cx37 on the downstream side. Surprisingly, Cx43 expression is strongly induced in the non-valve venous endothelium in superficial veins following wounding of the overlying skin. Moreover, we show that in Cx37-deficient mice, venous valves are entirely absent. Thus, Cx37, a protein involved in cell–cell communication, is one of only a few proteins identified so far as critical for the development or maintenance of venous valves. Because Cxs are necessary for the development of valves in lymphatic vessels as well, our results support the notion of common molecular pathways controlling valve development in veins and lymphatic vessels.

Recently, we reported that recovery of tissue perfusion in the ischemic hindlimb was reduced, inflammatory response increased, and survival of distal limb tissue compromised in connexin 40 (Cx40)-deficient (Cx40(-/-)) mice. Here we evaluate whether genotype-specific differences in tissue perfusion, native vascular density, arteriogenesis, blood pressure, and chronic ANG II type 1 receptor (AT1R) activation contribute to poor recovery of ischemic hindlimb tissue in Cx40(-/-) mice. Hindlimb ischemia was induced in wild-type (WT), Cx40(-/-), and losartan-treated Cx40(-/-) mice by using surgical procedures that either maintained (mild surgery) or compromised (severe surgery) perfusion of major collateral vessels supplying the distal limb. Pre- and postsurgical hindlimb perfusion was evaluated, and tissue survival, microvascular density, and macrophage infiltration were documented during recovery. Hindlimb perfusion was compromised in presurgical Cx40(-/-) versus WT mice despite comparable native microvascular density. Hindlimb perfusion 24 h postsurgery in Cx40(-/-) and WT mice was comparable after mild surgery (collateral vessels maintained), but compromised arteriogenesis in Cx40(-/-) animals nevertheless limited subsequent recovery of tissue perfusion and compromised tissue survival. Prolonged pre- and postsurgical treatment of Cx40(-/-) mice with losartan (an AT1R antagonist) normalized blood pressure but did not improve tissue perfusion or survival, despite reduced macrophage infiltration. Thus it appears Cx40 is necessary for normal tissue perfusion and for recovery of perfusion, arteriogenesis, and tissue survival in the ischemic hindlimb. Our data suggest that Cx40(-/-) mice are at significantly greater risk for poor recovery from ischemic insult due to compromised regulation of tissue perfusion, vascular remodeling, and prolonged inflammatory response.

The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor (37,43)Gap27 on Cx40(-/-) mouse platelets and of the Cx40 inhibitor (40)Gap27 on Cx37(-/-) mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.

The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37(-/-)) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40(-/-) animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37(-/-) animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37(-/-) mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37(-/-) animals compared with WT animals. Cx37(-/-) mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37(-/-) animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.