Interstate Highway Connections and Traced Gun Transfers Between the 48 Contiguous United States.

Importance: Interstate gun flow has critical implications for gun violence prevention, as gun transfers across state lines can undermine local gun control policies. Objective: To identify possible gun trafficking routes along interstate highways in the US. Design, Setting, and Participants: This repeated-measures, ecological, cross-sectional study used data from the Bureau of Alcohol, Tobacco, Firearms and Explosives from January 1, 2010, to December 31, 2019, to examine associations between interstate connections via 13 highways that each spanned at least 1000 miles and interstate traced gun transfer counts for the 48 contiguous United States. Analyses were completed in November 2023. Exposures: Characteristics of the origin states and the transportation connections between the destination state and the origin states. Main Outcomes and Measures: The main outcome was the total count of guns used in crimes in each destination state per year that were originally purchased in the origin state. Bayesian conditional autoregressive Poisson models were used to examine associations between the count of guns used in crime traced to interstate purchases and interstate highway connections between origin and destination states. Results: Between 2010 and 2019, 526 801 guns used in crimes in the contiguous 48 states were traced to interstate purchases. Northbound gun transfers along the Interstate 95 corridor were greater than expected to New Jersey (incidence rate ratio [IRR], 2.80; 95% credible interval [CrI], 1.01-7.68) and Maryland (IRR, 3.07; 95% CrI, 1.09-8.61); transfers were similarly greater along Interstate 15 southbound, Interstate 25 southbound, Interstate 35 southbound, Interstate 75 northbound and southbound, Interstate 10 westbound, and Interstate 20 eastbound and westbound. Conclusions and Relevance: This repeated-measures, ecological, cross-sectional study identified that guns used in crimes traced to interstate purchases moved routinely between states along multiple major transportation routes. Interstate gun transfers are a major contributor to gun crime, injury, and death in the US. National policies and interstate cooperation are needed to address this issue.

Defects in vein valve PROX1/FOXC2 antithrombotic pathway in endothelial cells drive the hypercoagulable state induced by trauma and critical illness.

OBJECTIVES: Deep venous thrombosis (DVT) causes significant morbidity and mortality after trauma. Recently, we have shown that blood flow patterns at vein valves induce oscillatory stress genes, which maintain an anticoagulant endothelial phenotype that inhibits spontaneous clotting at vein valves and sinuses, is lost in the presence of DVT in human pathological samples, and is dependent on expression of the transcription factor FOXC2. We describe an assay, modifying our mouse multiple injury system, which shows evidence of clinically relevant microthrombosis and hypercoagulability applicable to the study of spontaneous DVT in trauma without requiring direct vascular injury or ligation. Finally, we investigated whether these model findings are relevant to a human model of critical illness by examining gene expression changes by quantitative polymerase chain reaction and immunofluorescence in veins collected from critically ill. METHODS: C57/Bl6 mice were subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Serum was assayed for d-dimer at 2, 6, 24, and 48 hours after injury by enzyme-linked immunosorbent assay. For the thrombin clotting assay, veins of the leg were exposed, 100 µL of 1 mM rhodamine (6 g) was injected retro-orbitally, and 450 µg/mL thrombin was then applied to the surface of the vein with examination of real-time clot formation via in vivo immunofluorescence microscopy. Images were then examined for percentage area of clot coverage of visible mouse saphenous and common femoral vein. Vein valve specific knockout of FOXC2 was induced with tamoxifen treatment in PROX1 Ert2Cre FOXC2 fl/fl mice as previously described. Animals were then subjected to a modified mouse multiple injury model with liver crush injury, crush and pseudofracture of a single lower extremity, and 15% total blood volume hemorrhage. Twenty-four hours after injury, we examined the valve phenotype in naive versus multiple injury animals, with and without loss of the FOXC2 gene from the vein valve (FOXC2 del ) via the thrombin assay. Images were then examined for proximity of clot formation to the valve present at the junction of the mouse saphenous, tibial, and superficial femoral vein and presence of spontaneous microthrombi present in the veins before exposure to thrombin. Human vein samples were obtained from excess tissue preserved after harvest for elective cardiac surgery and from organ donors after organ procurement. Sections were submitted for paraffin embedding and then assayed by immunofluorescence for PROX1, FOXC2, thrombomodulin, endothelial protein C receptor, and von Willebrand's factor. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee, and all human studies reviewed and approved by the institutional review board. RESULTS: After mouse multiple injuries, enzyme-linked immunosorbent assay for d-dimer showed evidence of products of fibrin breakdown consistent with formation of clot related to injury, fibrinolysis, and/or microthrombosis. The thrombin clotting assay demonstrated higher percentage area of vein covered with clot when exposed to thrombin in the multiple injury animals compared with uninjured (45% vs. 27% p = 0.0002) consistent with a phenotype of hypercoagulable state after trauma in our model system. Unmanipulated FoxC2 knockout mice manifest increased clotting at the vein valve as compared with unmanipulated wild type animals. After multiple injuries, wild type mice manifest increase clotting at the vein after thrombin exposure ( p = 0.0033), and equivalent to that of valvular knockout of FoxC2 (FoxC2del), recapitulating the phenotype seen in FoxC2 knockout animals. The combination of multiple injuries and FoxC2 knockout resulted in spontaneous microthrombi in 50% of the animals, a phenotype not observed with either multiple injuries or FoxC2 deficiency alone (χ 2 , p = 0.017). Finally, human vein samples demonstrated the protective vein valve phenotype of increased FOXC2 and PROX1 and showed decreased expression in the critically ill organ donor population by immunofluorescence imaging in organ donor samples. CONCLUSION: We have established a novel model of posttrauma hypercoagulation that does not require direct restriction of venous flow or direct injury to the vessel endothelium to assay for hypercoagulability and can generate spontaneous microthrombosis when combined with valve-specific FOXC2 knockout. We find that multiple injuries induce a procoagulant phenotype that recapitulates the valvular hypercoagulability seen in FOXC2 knockout and, in critically ill human specimens, find evidence for loss of oscillatory shear stress-induced gene expression of FOXC2 and PROX1 in the valvular endothelium consistent with potential loss of DVT-protective valvular phenotype.

Extrathoracic multiple trauma dysregulates neutrophil function and exacerbates pneumonia-induced lung injury.

BACKGROUND: Forty percent of critically ill trauma patients will develop an infectious complication. Pneumonia is the most common cause of death of trauma patients surviving their initial insult. We previously demonstrated that polytrauma (PT), defined as two or more severe injuries in at least two areas of the body, induces emergency hematopoiesis characterized by accelerated myelopoiesis in the bone marrow and increased myeloid cell frequency in the peripheral tissues. We hypothesized that PT alone induces priming of neutrophils, resulting in hyperactivation upon secondary exposure to bacteria and causing acute lung injury and increased susceptibility to secondary exposure to Pseudomonas aeruginosa pneumonia. METHODS: C57BL/6 mice were subjected to PT consisting of a lower extremity pseudofracture, liver crush injury, and 15% blood-volume hemorrhage. Pneumonia was induced by intratracheal injection of 5 × 106 CFU live P. aeruginosa or 1 × 107 of heat-killed P. aeruginosa (HKPA). For reactive oxygen species (ROS), studies polymorphonuclear neutrophils (PMNs) were isolated by immunomagnetic bead negative selection and stimulated ex-vivo with HKPA. Reactive oxygen species production was measured by immunofluorescence. For histology, lung sections were stained by hematoxylin-eosin and analyzed by a blinded grader. RESULTS: Polytrauma induced persistent changes in immune function at baseline and to secondary infection. Pneumonia after injury resulted in increased mortality (60% vs. 5% p < 0.01). Blood neutrophils from PT mice had higher resting (unstimulated) ROS production than in naive animals (p < 0.02) demonstrating priming of the neutrophils following PT. After intratracheal HKPA injection, bronchoalveolar lavage PMNs from injured mice had higher ROS production compared with naive mice (p < 0.01), demonstrating an overexuberant immunopathologic response of neutrophils following PT. CONCLUSION: Polytrauma primes neutrophils and causes immunopathologic PMN ROS production, increased lung injury and susceptibility to secondary bacterial pneumonia. These results suggest that trauma-induced immune dysfunction can cause immunopathologic response to secondary infection and suggests neutrophil-mediated pulmonary damage as a therapeutic target for posttrauma pneumonia.

Universal background checks for handgun purchases can reduce homicide rates of African Americans.

BACKGROUND: Federal law requires background checks for firearms purchased from licensed dealers, but states can extend requirements to private sales of handguns and purchases at gun shows (universal background checks for handguns [UBC-HG]). Although firearm homicide disproportionately affects African Americans, little is known about how UBG-HG impacts African Americans. We hypothesized that implementation of UBC-HG would reduce rates of firearm homicide of African Americans. METHODS: We collected Centers for Disease Control firearm homicide counts for African American and white populations in the 50 states, 1999 to 2017. Laws were drawn from the State Firearm Laws Database. The exposure and outcome of interest were UBC-HG adoption and firearm homicide. We included non-Hispanic African American and non-Hispanic white populations. We used Poisson regression to perform a differences-in-differences analysis. A categorical variable for state accounted for time-stable state characteristics. We controlled for year to account for trends over time unrelated to policy. We controlled for state-specific, time-variable factors, including median household income, population younger than 25 years or 65 years or older, alcohol consumption, and count of firearm laws (UBC-HG excluded). Standard errors were adjusted for clustering at the state level. RESULTS: The firearm homicide rate among whites was 1.8 per 100,000 (interquartile range, 1.2-2.7) ranging from 1.4 in 2011 to 1.8 in 2016. The firearm homicide rate was 15.6 per 100,000 (interquartile range, 11.6-21.0) among African Americans, ranging from 14.0 in 2009 to 19.6 in 2017. While no significant difference in firearm homicides among whites (incidence rate ratio, 0.93; 95% confidence interval, 0.73-1.20) was appreciated, the passage of UBC-HG was associated with an 19% decrease in African Americans firearm homicides (incidence rate ratio, 0.81; 95% confidence interval, 0.70-0.94; p = 0.006). CONCLUSION: Implementing UBC-HG was associated with decreased firearm homicides among African Americans-the population most at risk. Expanding UBC-HG may be an effective approach to reducing racial disparities in firearm homicides. LEVEL OF EVIDENCE: Epidemiological, level III.

Firearm laws and illegal firearm flow between US states.

BACKGROUND: Considerable variation in firearm legislation exists. Prior studies show an association between stronger state laws and fewer firearm deaths. We hypothesized that firearms would flow from states with weaker laws to states with stronger laws based on proximity and population. METHODS: Crime gun trace data from 2015 to 2017 was accessed from the Bureau of Alcohol, Tobacco, Firearms and Explosives and compared with the count and composition of firearm legislation in 2015 among the contiguous 48 states. Additional independent variables included population, median household income, distance, and presence or absence of a shared border. We used Exponential Random Graph Models to identify predictors of traced firearm transfers between origin and destination states. RESULTS: After controlling for network structure, firearm laws in origin states were associated with fewer traced firearm transfers (incidence rate ratio [IRR], 0.88; 95% confidence interval [CI], 0.83-0.93; p < 0.001). Conversely, more firearm laws in destination states were associated with more traced firearm transfers (IRR, 1.10; 95% CI, 1.06-1.15; p < 0.001). Larger population at the origin was associated with increased transfers (IRR, 1.38; 95%CI, 1.27-1.50; p < 0.001), as was larger population at the destination state (IRR, 1.45; 95% CI, 1.35-1.56; p < 0.001). Greater distance was associated with fewer transfers (for each 1,000 km; IRR, 0.35; 95% CI, 0.27-0.46; p < 0.001), and transfers were greater between adjacent states (IRR, 2.49; 95% CI, 1.90-3.27; p < 0.001). CONCLUSION: State firearm legislation has a significant impact on gun trafficking even after controlling for network structure. States with stricter firearm legislation are negatively impacted by states with weaker regulations, as crime guns flow from out-of-state. LEVEL OF EVIDENCE: Epidemiologic, level III.

Hemodynamic regulation of perivalvular endothelial gene expression prevents deep venous thrombosis.

Deep venous thrombosis (DVT) and secondary pulmonary embolism cause approximately 100,000 deaths per year in the United States. Physical immobility is the most significant risk factor for DVT, but a molecular and cellular basis for this link has not been defined. We found that the endothelial cells surrounding the venous valve, where DVTs originate, express high levels of FOXC2 and PROX1, transcription factors known to be activated by oscillatory shear stress. The perivalvular venous endothelial cells exhibited a powerful antithrombotic phenotype characterized by low levels of the prothrombotic proteins vWF, P-selectin, and ICAM1 and high levels of the antithrombotic proteins thrombomodulin (THBD), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). The perivalvular antithrombotic phenotype was lost following genetic deletion of FOXC2 or femoral artery ligation to reduce venous flow in mice, and at the site of origin of human DVT associated with fatal pulmonary embolism. Oscillatory blood flow was detected at perivalvular sites in human veins following muscular activity, but not in the immobile state or after activation of an intermittent compression device designed to prevent DVT. These findings support a mechanism of DVT pathogenesis in which loss of muscular activity results in loss of oscillatory shear-dependent transcriptional and antithrombotic phenotypes in perivalvular venous endothelial cells, and suggest that prevention of DVT and pulmonary embolism may be improved by mechanical devices specifically designed to restore perivalvular oscillatory flow.

Myeloid p53 regulates macrophage polarization and venous thrombus resolution by inflammatory vascular remodeling in mice.

Deep venous thrombosis (DVT) remains a common and serious cardiovascular problem with both fatal and long-term consequences. The consequences of DVT include the development of postthrombotic syndrome in 25% to 60% of DVT patients. Despite the clinical importance of venous thrombus resolution, the cellular and molecular mediators involved are poorly understood, and currently there is no molecular therapy to accelerate this process. Several lines of evidence suggest that a complex and interrelated array of molecular signaling processes are involved in the inflammatory vascular remodeling associated with the resolution of DVT. Here, we have identified a role for the tumor suppressor gene p53 in regulating venous thrombus resolution. Using the stasis model of venous thrombosis and resolution in mice, we found that genetic deficiency of p53 or pharmacologic inhibition by pifithrin impairs thrombus resolution and is associated with increased fibrosis and altered expression of matrix metalloproteinase-2. The effect of p53 loss was mediated by cells of the myeloid lineage, resulting in enhanced polarization of the cytokine milieu toward an M1-like phenotype. Furthermore, augmentation of p53 activity using the pharmacological agonist of p53, quinacrine, accelerates venous thrombus resolution in a p53-dependent manner, even after establishment of thrombosis. Together, these studies define mechanisms by which p53 regulates thrombus resolution by increasing inflammatory vascular remodeling of venous thrombi in vivo, and the potential therapeutic application of a p53 agonist as a treatment to accelerate this process in patients with DVT.

Recanalization and flow regulate venous thrombus resolution and matrix metalloproteinase expression in vivo.

OBJECTIVE: We examined the role of thrombus recanalization and ongoing blood flow in the process of thrombus resolution by comparing two murine in vivo models of deep venous thrombosis. METHODS: In CD1 mice, we performed surgical inferior vena cava ligation (stasis thrombosis), stenosis (thrombosis with recanalization), or sham procedure. We analyzed thrombus weight over time as a measure of thrombus resolution and quantified the messenger RNA and protein levels of membrane-type matrix metalloproteinases (MT-MMPs) as well as effectors of the plasmin complex at days 4, 8, and 12 after surgery. RESULTS: Despite similar initial thrombus size, the presence of ongoing blood flow (stenosis model) was associated with a 45.91% subsequent improvement in thrombus resolution at day 8 and 12.57% at day 12 compared with stasis thrombosis (ligation model). Immunoblot and real-time polymerase chain reaction analysis demonstrated a difference in MMP-2 and MMP-9 activity at day 8 between the two models (P = .03 and P = .006, respectively) as well as a difference in MT2-MMP gene expression at day 8 (P = .044) and day 12 (P = .03) and MT1-MMP protein expression at day 4 (P = .021). Histologic analyses revealed distinct areas of recanalization in the thrombi of the stenosis model compared with the ligation model as well as the recruitment of inflammatory cells, especially macrophages, and a focal pattern of localized expression of MT1-MMP and MT3-MMP proteins surrounding the areas of recanalization in the stenosis model. CONCLUSIONS: Recanalization and ongoing blood flow accelerate deep venous thrombus resolution in vivo and are associated with distinct patterns of MT1-MMP and MT3-MMP expression and macrophage localization in areas of intrathrombus recanalization.

Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells.

The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.

Kruppel-like factor 4 abrogates myocardin-induced activation of smooth muscle gene expression.

Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC.

Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells.

Knockout of transforming growth factor (TGF)-beta1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-beta1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle alpha-actin (SMalphaA), SM22alpha, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-beta with an adenovirus expressing a soluble truncated TGF-beta type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-beta1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-beta-Smad signaling for an early vs. late SMC marker gene in that SMalphaA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-beta1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

Lost in transdifferentiation.

What are the true origins of the smooth muscle cells (SMCs) present in the intimal lesions of transplant arteriosclerosis? A new study in the JCI shows that Sca-1(+) cells purified from the mouse aortic root can migrate through an irradiated vein graft to the neointima of the vessel and transdifferentiate to express the early SMC differentiation marker gene SM22. Do Sca-1(+) cells transdifferentiate into SMC-like cells, or is activation of SMC marker genes a consequence of fusion of these cells with preexisting SMCs, a possibility raised by results of studies of adult stem cells in animal models of liver regeneration ? Or could this be bona fide transdifferentiation that recapitulates the pathologic processes in humans?

Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes.

The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM alpha-actin, SM-myosin heavy chain, and SM22alpha by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.