Cyclooxygenase-2 deficiency attenuates lipopolysaccharide-induced inflammation, apoptosis, and acute lung injury in adult mice.

Many lung diseases are caused by an excessive inflammatory response, and inflammatory lung diseases are often modeled using lipopolysaccharide (LPS) in mice. Cyclooxygenase-2 (COX-2) encoded by the Ptgs2 gene is induced in response to inflammatory stimuli including LPS. The objective of this study was to test the hypothesis that mice deficient in COX-2 (Ptgs2-/-) will be protected from LPS-induced lung injury. Wild-type (WT; CD1 mice) and Ptgs2-/- mice (on a CD1 background) were treated with LPS or vehicle for 24 h. LPS treatment resulted in histological evidence of lung injury, which was attenuated in the Ptgs2-/- mice. LPS treatment increased the mRNA levels for tumor necrosis factor-α, interleukin-10, and monocyte chemoattractant protein-1 in the lungs of WT mice, and the LPS-induced increases in these levels were attenuated in the Ptgs2-/- mice. The protein levels of active caspase-3 and caspase-9 were lower in the LPS-treated lungs of Ptgs2-/- mice than in LPS-treated WT mice, as were the number of terminal deoxynucleotide transferase dUTP nick end labeling-positive cells in lung sections. LPS exposure resulted in a greater lung wet-to-dry weight ratio (W/D) in WT mice, suggestive of pulmonary edema, while in LPS-treated Ptgs2-/- mice, the W/D was not different from controls and less than in LPS-treated WT mice. These results demonstrate that COX-2 is involved in the inflammatory response to LPS and suggest that COX-2 not only acts as a downstream participant in the inflammatory response, but also acts as a regulator of the inflammatory response likely through a feed-forward mechanism following LPS stimulation.

Patent Ductus Arteriosus of the Preterm Infant.

Postnatal ductal closure is stimulated by rising oxygen tension and withdrawal of vasodilatory mediators (prostaglandins, nitric oxide, adenosine) and by vasoconstrictors (endothelin-1, catecholamines, contractile prostanoids), ion channels, calcium flux, platelets, morphologic maturity, and a favorable genetic predisposition. A persistently patent ductus arteriosus (PDA) in preterm infants can have clinical consequences. Decreasing pulmonary vascular resistance, especially in extremely low gestational age newborns, increases left-to-right shunting through the ductus and increases pulmonary blood flow further, leading to interstitial pulmonary edema and volume load to the left heart. Potential consequences of left-to-right shunting via a hemodynamically significant patent ductus arteriosus (hsPDA) include increased risk for prolonged ventilation, bronchopulmonary dysplasia, necrotizing enterocolitis or focal intestinal perforation, intraventricular hemorrhage, and death. In the last decade, there has been a trend toward less aggressive treatment of PDA in preterm infants. However, there is a subgroup of infants who will likely benefit from intervention, be it pharmacologic, interventional, or surgical: (1) prophylactic intravenous indomethacin in highly selected extremely low gestational age newborns with PDA (<26 + 0/7 weeks' gestation, <750 g birth weight), (2) early targeted therapy of PDA in selected preterm infants at particular high risk for PDA-associated complications, and (3) PDA ligation, catheter intervention, or oral paracetamol may be considered as rescue options for hsPDA closure. The impact of catheter-based closure of hsPDA on clinical outcomes should be determined in future prospective studies. Finally, we provide a novel treatment algorithm for PDA in preterm infants that integrates the several treatment modalities in a staged approach.

Prostaglandin-Endoperoxide Synthase 1 Mediates the Timing of Parturition in Mice Despite Unhindered Uterine Contractility.

Cyclooxygenase (COX)-derived prostaglandins stimulate uterine contractions and prepare the cervix for parturition. Prior reports suggest Cox-1 knockout (KO) mice exhibit delayed parturition due to impaired luteolysis, yet the mechanism for late-onset delivery remains unclear. Here, we examined key factors for normal onset of parturition to determine whether any could account for the delayed parturition phenotype. Pregnant Cox-1KO mice did not display altered timing of embryo implantation or postimplantation growth. Although messenger RNAs of contraction-associated proteins (CAPs) were differentially expressed between Cox-1KO and wild-type (WT) myometrium, there were no differences in CAP agonist-induced intracellular calcium release, spontaneous or oxytocin (OT)-induced ex vivo uterine contractility, or in vivo uterine contractile pressure. Delayed parturition in Cox-1KO mice persisted despite exogenous OT treatment. Progesterone (P4) withdrawal, by ovariectomy or administration of the P4-antagonist RU486, diminished the delayed parturition phenotype of Cox-1KO mice. Because antepartum P4 levels do not decline in Cox-1KO females, P4-treated WT mice were examined for the effect of this hormone on in vivo uterine contractility and ex vivo cervical dilation. P4-treated WT mice had delayed parturition but normal uterine contractility. Cervical distensibility was decreased in Cox-1KO mice on the day of expected delivery and reduced in WT mice with long-term P4 treatment. Collectively, these findings show that delayed parturition in Cox-1KO mice is the result of impaired luteolysis and cervical dilation, despite the presence of strong uterine contractions.

Erythematous plaques and papules on a premature infant.

A 2240 gram boy was born at 33.2 weeks gestation with nonblanching, deeply erythematous plaques and papules on the back, flanks, and scalp (Figure 1). His mother was GBS positive and on antibiotic suppression for prior cutaneous MRSA and urinary tract infections. Intrapartum intravenous Penicillin G was administered, and the amniotic sac was artificially ruptured 4 hours prior to delivery to facilitate labor. The delivery was uncomplicated without concern for chorioamnionitis, but the patient initially required CPAP for respiratory distress with 1-minute and 5-minute Apgar scores of 7 and 8, respectively. A skin punch biopsy is shown (Figure 2).

Selective serotonin reuptake inhibitor exposure constricts the mouse ductus arteriosus in utero.

Use of selective serotonin reuptake inhibitors (SSRIs) is common during pregnancy. Fetal exposure to SSRIs is associated with persistent pulmonary hypertension of the newborn (PPHN); however, a direct link between the two has yet to be established. Conversely, it is well known that PPHN can be caused by premature constriction of the ductus arteriosus (DA), a fetal vessel connecting the pulmonary and systemic circulations. We hypothesized that SSRIs could induce in utero DA constriction. Using isolated vessels and whole-animal models, we sought to determine the effects of two commonly prescribed SSRIs, fluoxetine and sertraline, on the fetal mouse DA. Cannulated vessel myography studies demonstrated that SSRIs caused concentration-dependent DA constriction and made vessels less sensitive to prostaglandin-induced dilation. Moreover, in vivo studies showed that SSRI-exposed mice had inappropriate DA constriction in utero. Taken together, these findings establish that SSRIs promote fetal DA constriction and provide a potential mechanism by which SSRIs could contribute to PPHN.

Competition and allostery govern substrate selectivity of cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Effects of Advancing Gestation and Non-Caucasian Race on Ductus Arteriosus Gene Expression.

OBJECTIVE: To identify genes affected by advancing gestation and racial/ethnic origin in human ductus arteriosus (DA). STUDY DESIGN: We collected 3 sets of DA tissue (n = 93, n = 89, n = 91; total = 273 fetuses) from second trimester pregnancies. We examined four genes, with DNA polymorphisms that distribute along racial lines, to identify "Caucasian" and "non-Caucasian" DA. We used real time polymerase chain reaction to measure RNA expression of 48 candidate genes involved in functional closure of the DA, and used multivariable regression analyses to examine the relationships between advancing gestation, "non-Caucasian" race, and gene expression. RESULTS: Mature gestation and non-Caucasian race are significant predictors for identifying infants who will close their patent DA when treated with indomethacin. Advancing gestation consistently altered gene expression in pathways involved with oxygen-induced constriction (eg, calcium-channels, potassium-channels, and endothelin signaling), contractile protein maturation, tissue remodeling, and prostaglandin and nitric oxide signaling in all 3 tissue sets. None of the pathways involved with oxygen-induced constriction appeared to be altered in "non-Caucasian" DA. Two genes, SLCO2A1 and NOS3, (involved with prostaglandin reuptake/metabolism and nitric oxide production, respectively) were consistently decreased in "non-Caucasian" DA. CONCLUSIONS: Prostaglandins and nitric oxide are the most important vasodilators opposing DA closure. Indomethacin inhibits prostaglandin production, but not nitric oxide production. Because decreased SLCO2A1 and NOS3 expression can lead to increased prostaglandin and decreased nitric oxide concentrations, we speculate that prostaglandin-mediated vasodilation may play a more dominant role in maintaining the "non-Caucasian" patent DA, making it more likely to close when inhibited by indomethacin.

Cross-species transcriptomic approach reveals genes in hamster implantation sites.

The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS.

Aminoglycoside-mediated relaxation of the ductus arteriosus in sepsis-associated PDA.

Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, <25 wk and >32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA.

Transcriptional profiling reveals ductus arteriosus-specific genes that regulate vascular tone.

Failure of the ductus arteriosus (DA) to close at birth can lead to serious complications. Conversely, certain profound congenital cardiac malformations require the DA to be patent until corrective surgery can be performed. In each instance, clinicians have a very limited repertoire of therapeutic options at their disposal - indomethacin or ibuprofen to close a patent DA (PDA) and prostaglandin E1 to maintain patency of the DA. Neither treatment is specific to the DA and both may have deleterious off-target effects. Therefore, more therapeutic options specifically targeted to the DA should be considered. We hypothesized the DA possesses a unique genetic signature that would set it apart from other vessels. A microarray was used to compare the genetic profiles of the murine DA and ascending aorta (AO). Over 4,000 genes were differentially expressed between these vessels including a subset of ion channel-related genes. Specifically, the alpha and beta subunits of large-conductance calcium-activated potassium (BKCa) channels are enriched in the DA. Gain- and loss-of-function studies showed inhibition of BKCa channels caused the DA to constrict, while activation caused DA relaxation even in the presence of O2. This study identifies subsets of genes that are enriched in the DA that may be used to develop DA-specific drugs. Ion channels that regulate DA tone, including BKCa channels, are promising targets. Specifically, BKCa channel agonists like NS1619 maintain DA patency even in the presence of O2 and may be clinically useful.

Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization, and defense against bacterial endotoxin in hamsters.

Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types and their hormonal regulation, cell type, and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5, and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone (P4) is the major uterine Akp2 inducer, both P4 and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. By contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection.

Enteral feeding during indomethacin and ibuprofen treatment of a patent ductus arteriosus.

OBJECTIVE: To test the hypothesis that infants who are just being introduced to enteral feedings will advance to full enteral nutrition at a faster rate if they receive "trophic" (15 mL/kg/d) enteral feedings while receiving indomethacin or ibuprofen treatment for patent ductus arteriosus. STUDY DESIGN: Infants were eligible for the study if they were 23(1/7)-30(6/7) weeks' gestation, weighed 401-1250 g at birth, received maximum enteral volumes ≤60 mL/kg/d, and were about to be treated with indomethacin or ibuprofen. A standardized "feeding advance regimen" and guidelines for managing feeding intolerance were followed at each site (N = 13). RESULTS: Infants (N = 177, 26.3 ± 1.9 weeks' mean ± SD gestation) were randomized at 6.5 ± 3.9 days to receive "trophic" feeds ("feeding" group, n = 81: indomethacin 80%, ibuprofen 20%) or no feeds ("fasting [nil per os]" group, n = 96: indomethacin 75%, ibuprofen 25%) during the drug administration period. Maximum daily enteral volumes before study entry were 14 ± 15 mL/kg/d. After drug treatment, infants randomized to the "feeding" arm required fewer days to reach the study's feeding volume end point (120 mL/kg/d). Although the enteral feeding end point was reached at an earlier postnatal age, the age at which central venous lines were removed did not differ between the 2 groups. There were no differences between the 2 groups in the incidence of infection, necrotizing enterocolitis, spontaneous intestinal perforation, or other neonatal morbidities. CONCLUSION: Infants required less time to reach the feeding volume end point if they were given "trophic" enteral feedings when they received indomethacin or ibuprofen treatments.

Omental grafting: a cell-based therapy for blood vessel repair.

Clinicians regularly transplant omental pedicles to repair a wide variety of injured tissues, but the basic mechanism underlying this efficacious procedure is not understood. One possibility that has not been addressed is the ability of omentum to directly contribute regenerative cells to injured tissues. We hypothesized that if omental progenitor cells could be mobilized to incorporate into damaged tissue, the power of this therapy would be greatly expanded. Labelled omental grafts were transplanted into a murine carotid artery injury model. Selected grafts were treated with thymosin ß4 (Tß4) prior to transplantation to investigate the effects of chemical potentiation on healing. We found treatment of grafts with Tß4-induced progenitor cells to fully integrate into the wall of injured vessels and differentiate into vascular smooth muscle. Myographic studies determined that arteries receiving Tß4-stimulated grafts were functionally indistinguishable from uninjured controls. Concurrent in vitro analyses showed that Tß4 promoted proliferation, migration and trans-differentiation of cells via AKT signalling. This study is the first to demonstrate that omentum can provide progenitor cells for repair, thus revealing a novel and naturally occurring source of vascular smooth muscle for use in cell-based therapies. Furthermore, our data show that this system can be optimized with inducing factors, highlighting a more powerful therapeutic potential than that of its current clinical application. This is a paradigm-setting concept that lays the foundation for the use of chemical genetics to enhance therapeutic outcomes in a myriad of fields.

Unexpected extra-renal effects of loop diuretics in the preterm neonate.

UNLABELLED: The loop diuretics furosemide and bumetanide are commonly used in neonatal intensive care units (NICUs). Furosemide, because of its actions on the ubiquitous Na(+) -K(+) -2Cl(-) isoform cotransporter and its promotion of prostanoid production and release, also has non-diuretic effects on vascular smooth muscle, airways, the ductus arteriosus and theoretically the gastrointestinal tract. Loop diuretics also affect the central nervous system through modulation of the GABA-A chloride channel. CONCLUSION: The loop diuretics have a variety of biological effects that are potentially harmful as well as beneficial. Care should be taken with the use of these agents because the range of their effects may be broader than the single action sought by the prescribing physician.

Overexpression of angiopoietin-2 impairs myocardial angiogenesis and exacerbates cardiac fibrosis in the diabetic db/db mouse model.

The angiopoietins/Tie-2 system is essential for the maintenance of vascular integrity and angiogenesis. The functional role of angiopoietin-2 (Ang-2) in the regulation of angiogenesis is dependent on other growth factors such as VEGF and a given physiopathological conditions. This study investigates the potential role of Ang-2 in myocardial angiogenesis and fibrosis formation in the diabetic db/db mouse. Diabetic db/db mice received intramyocardial administration of either adenovirus Ang-2 (Ad-CMV-Ang-2) or Ad-ß-gal. The levels of Tie-2, VEGF, caspase-3, Wnt7b, fibroblast-specific protein-1 (FSP-1), and adhesion molecules (ICAM-1 and VCAM-1) expression were measured. Apoptosis, capillary density, and cardiac fibrosis were also analyzed in the db/db mouse hearts. Overexpression of Ang-2 suppressed Tie-2 and VEGF expression in db/db mouse hearts together with significant upregulation of Wnt7b expression. Overexpression of Ang-2 also sensitizes ICAM-1 and VCAM-1 expression in db/db mouse hearts. Immunohistochemical analysis revealed that overexpression of Ang-2 resulted in a gradual apoptosis as well as interstitial fibrosis formation, these leading to a significant loss of capillary density. Data from these studies were confirmed in cultured mouse heart microvascular endothelial cells (MHMEC) exposed to excessive Ang-2. Exposure of MHMEC to Ang-2 resulted in increased caspase-3 activity and endothelial apoptosis. Knockdown of Ang-2 attenuated high glucose-induced endothelial cell apoptosis. Further, counterbalance of Ang-2 by overexpression of Ang-1 reversed loss of capillary density and fibrosis formation in db/db mouse hearts. Our data demonstrate that Ang-2 increases endothelial apoptosis, sensitizes myocardial microvascular inflammation, and promotes cardiac fibrosis and thus contributes to loss of capillary density in diabetic diseases.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Regulation of the atheroma-enriched protein, SPRR3, in vascular smooth muscle cells through cyclic strain is dependent on integrin alpha1beta1/collagen interaction.

Atherosclerotic plaques express high levels of small proline-rich repeat protein (SPRR3), a previously characterized component of the cornified cell envelope of stratified epithelia, where it is believed to play a role in cellular adaptation to biomechanical stress. We investigated the physiological signals and underlying mechanism(s) that regulate atheroma-enriched SPRR3 expression in vascular smooth muscle cells (VSMCs). We showed that SPRR3 is expressed by VSMCs in both human and mouse atheromas. In cultured arterial VSMCs, mechanical cyclic strain, but neither shear stress nor lipid loading induced SPRR3 expression. Furthermore, this upregulation of SPRR3 expression was dependent on VSMC adherence to type I collagen. To link the mechanoregulation of SPRR3 to specific collagen/integrin interactions, we used blocking antibodies against either integrin alpha1 or alpha2 subunits and VSMCs from mice that lack specific collagen receptors. Our results showed a dependence on the alpha1beta1 integrin for SPRR3 expression induced by cyclic strain. Furthermore, we showed that integrin alpha1 but not alpha2 subunits were expressed on VSMCs within mouse lesions but not in normal arteries. Therefore, we identified the enrichment of the mechanical strain-regulated protein SPRR3 in VSMCs of both human and mouse atherosclerotic lesions whose expression is dependent on the collagen-binding integrin alpha1beta1 on VSMCs. These data suggest that SPRR3 may play a role in VSMC adaptation to local biomechanical stress within the plaque microenvironment.