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Cantú syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by gain-of-function (GoF) variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels and is characterized by low systemic vascular resistance, as well as tortuous, dilated, vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell autonomously within vascular smooth muscle cells (VSMCs) or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Whole-cell voltage clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild-type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were similar to those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs and suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF. The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs. Graphical Abstract [ABSTRACT FROM AUTHOR]

Cantu syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) [K.sub.ATP] channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knockin mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved by genetic downregulation of [K.sub.ATP] channel activity specifically in VSM, and by chronic administration of the clinically used [K.sub.ATP] channel inhibitor, glibenclamide. These findings demonstrate that VSM [K.sub.ATP] channel GoF underlies CS cardiac enlargement and that CS-associated abnormalities are reversible, and provide evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS., Introduction Cantu syndrome (CS) is a complex disorder with multiple cardiovascular abnormalities, including edema, dilated and tortuous blood vessels with decreased systemic vascular resistance, patent ductus arteriosus (PDA), and marked [...]

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration. [ABSTRACT FROM AUTHOR]

Thoracic aortic aneurysm is characterized by dilation of the aortic diameter by greater than 50%, which can lead to dissection or rupture. Common histopathology includes extracellular matrix remodeling that may affect transmural mass transport, defined as the movement of fluids and solutes across the wall. We measured in vitro ascending thoracic aorta mass transport in a mouse model with partial aneurysm phenotype penetration due to a mutation in the extracellular matrix protein fibulin-4 [Fbln4E57K/E57K, referred to as MU-A (aneurysm) or MU-NA (no aneurysm)]. To push the aneurysm phenotype, we also included MU mice with reduced levels of lysyl oxidase [Fbln4E57K/E57K;Lox + /-, referred to as MU-XA (extreme aneurysm)] and compared all groups to wild-type (WT) littermates. The phenotype variation allows investigation of how aneurysm severity correlates with mass transport parameters and extracellular matrix organization. We found that MU-NA ascending thoracic aortae have similar hydraulic conductance (Lp) to WT, but 397% higher solute permeability (x) for 4 kDa FITC-dextran. In contrast, MU-A and MU-XA ascending thoracic aortae have 44–68% lower Lp and similar x to WT. The results suggest that ascending thoracic aortic aneurysm progression involves an initial increase in x, followed by a decrease in Lp after the aneurysm has formed. All MU ascending thoracic aortae are longer and have increased elastic fiber fragmentation in the extracellular matrix. There is a negative correlation between diameter and Lp or x in MU ascending thoracic aortae. Changes in mass transport due to elastic fiber fragmentation could contribute to aneurysm progression or be leveraged for treatment. NEW & NOTEWORTHY Transmural mass transport is quantified in the ascending thoracic aorta of mice with a mutation in fibulin-4 that is associated with thoracic aortic aneurysms. Fluid and solute transport depend on aneurysm severity, correlate with elastic fiber fragmentation, and may be affected by proteoglycan deposition. Transport properties of the ascending thoracic aorta are provided and can be used in computational models. The changes in mass transport may contribute to aneurysm progression or be leveraged for aneurysm treatment. [ABSTRACT FROM AUTHOR]

RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell–specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell–specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell–specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion. ZFP36 proteins: The same, but different RNA binding proteins are an essential component of the regulatory machinery that prevents excessive lymphocyte activation by limiting target transcript translation or promoting mRNA decay. Using a series of conditional knockout mice, Cook et al. systematically investigated the role of the zinc finger protein 36 (ZFP36) family of RNA binding proteins in T cell homeostasis and autoimmunity. T cell–specific deletion of all three Zfp36 genes resulted in lethal, multiorgan inflammation accompanied by excessive production of IFN-γ and TNF, whereas mice deficient in any two family members were protected. Although ZFP36 proteins were redundant in controlling T cells during homeostasis, mice with T cells lacking Zfp36l1 and Zfp36l2 were resistant to development of experimental autoimmune encephalomyelitits, demonstrating that ZFP36 family members can also perform context-specific functions during autoimmunity. [ABSTRACT FROM AUTHOR]

DeMyctifying the tumor microenvironment: Anti-angiogenic treatments have so far delivered only modest success in patients with cancer, and the interactions between the tumor microenvironment and immunotherapies need to be better understood. Kabir et al. identified Myct1 as a critical factor for tumor growth and progression through dual effects on vascular development and tumor immunity. MYCT1 interacted with Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, and deficiency promoted antitumor T cell and macrophage phenotypes. The combination of Myct1 inhibition and immunotherapy led to tumor regression and long-term survival in tumor-bearing mice, suggesting that MYCT1 may be a promising target for antitumor therapy in the future. The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti–vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that Myct1, a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, Myct1 deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant (P < 0.05) correlation between MYCT1 expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased FOXP3 expression and increased antitumor macrophages in patients with low MYCT1 expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. Myct1-deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. Myct1 targeting combined with anti-PD1 treatment significantly (P < 0.05) increased complete tumor regression and long-term survival in anti-PD1–responsive and –refractory tumor models in mice. Our data collectively support a critical role for Myct1 in controlling tumor angiogenesis and reprogramming tumor immunity. Myct1-targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy. [ABSTRACT FROM AUTHOR]

Key points: We report that a sodium‐activated potassium current, IKNa, has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells.IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice.The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild‐type with raised blood pressure when challenged with vasoconstrictive agents.IKNa is negatively regulated by angiotensin II acting through Gαq protein‐coupled receptors.In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+‐dependent action potentials. Although several potassium currents have been reported to play a role in arterial smooth muscle (ASM), we find that one of the largest contributors to membrane conductance in both conduit and resistance ASMs has been inadvertently overlooked. In the present study, we show that IKNa, a sodium‐activated potassium current, contributes a major portion of macroscopic outward current in a critical physiological voltage range that determines intrinsic cell excitability; IKNa is the largest contributor to ASM cell resting conductance. A genetic knockout (KO) mouse strain lacking KNa channels (KCNT1 and KCNT2) shows only a modest hypertensive phenotype. However, acute administration of vasoconstrictive agents such as angiotensin II (Ang II) and phenylephrine results in an abnormally large increase in blood pressure in the KO animals. In wild‐type animals Ang II acting through Gαq protein‐coupled receptors down‐regulates IKNa, which increases the excitability of the ASMs. The complete genetic removal of IKNa in KO mice makes the mutant animal more vulnerable to vasoconstrictive agents, thus producing a paroxysmal‐hypertensive phenotype. This may result from the lowering of cell resting K+ conductance allowing the cells to depolarize more readily to a variety of excitable stimuli. Thus, the sodium‐activated potassium current may serve to moderate blood pressure in instances of heightened stress. IKNa may represent a new therapeutic target for hypertension and stroke. Key points: We report that a sodium‐activated potassium current, IKNa, has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells.IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice.The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild‐type with raised blood pressure when challenged with vasoconstrictive agents.IKNa is negatively regulated by angiotensin II acting through Gαq protein‐coupled receptors.In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+‐dependent action potentials. [ABSTRACT FROM AUTHOR]

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain. [ABSTRACT FROM AUTHOR]

We describe the successful treatment of a pediatric transplant patient with simultaneous intermittent hemodialysis and therapeutic plasma exchange (TPE). The patient presented with kidney graft failure. He had life threatening electrolyte disturbances and fluid overload due to antibody-mediated rejection. Therefore, he was in need of both emergent kidney replacement therapy and TPE. Both extracorporeal circuits were set up, established, and maintained safely and effectively without difficulty or alarms. Running intermittent hemodialysis and TPE simultaneously significantly reduced therapy time, allowed both needed therapies priority, and provided a superior pediatric patient experience in an acute situation. [ABSTRACT FROM AUTHOR]

Increased arterial stiffness is a common characteristic of humans with Williams-Beuren syndrome and mouse models of elastin insufficiency. Arterial stiffness is associated with multiple negative cardiovascular outcomes, including myocardial infarction, stroke, and sudden death. Therefore, identifying therapeutic interventions that improve arterial stiffness in response to changes in elastin levels is of vital importance. The goal of this study was to determine the effect of chronic pharmacologic therapy with different classes of antihypertensive medications on arterial stiffness in elastin insufficiency. Elastin-insufficient mice 4-6 wk of age and wild-type littermates were subcutaneously implanted with osmotic micropumps delivering a continuous dose of one of the following: vehicle, losartan, nicardipine, or propranolol for 8 wk. At the end of treatment period, arterial blood pressure and large artery compliance and remodeling were assessed. Our results show that losartan and nicardipine treatment lowered blood pressure and pulse pressure in elastin-insufficient mice. Elastin and collagen content of abdominal aortas as well as ascending aorta and carotid artery biomechanics were not affected by any of the drug treatments in either genotype. By reducing pulse pressure and shifting the working pressure range of an artery to a more compliant region of the pressurediameter curve, antihypertensive medications may mitigate the consequences of arterial stiffness, an effect that is drug class independent. These data emphasize the importance of early recognition and longterm management of hypertension in Williams-Beuren syndrome and elastin insufficiency. [ABSTRACT FROM AUTHOR]

The article presents a case study of 16-year-old adolescent female presenting with seizures and renal failure due to a hypertensive crisis. Her kidny function improved dur to blood pressure treatment and imaging identified moyamoya disease. This case highlights that moyamoya must be considered in the differential diagnosis of patients presenting with hypertensive emergency and renal failure.

The article presents a study which uses a new transgenic mouse model with human (h) angiotensin converting enzyme type 2 (ACE2) to clarify the role of central ACE2 under the control of synapsin promoter. It states that overactive renin-angiotensin system might activate the ACE2, which is a new member of the brain-angiotensin system. It cites that the results showed neurogenic hypertension is attenuated by the brain-selective overexpression of (h) ACE2.

The article provides information on the endothelial function in transgenic mice which expresses dominant negative mutants of PPARγ that plays a protective role in the vasculature. Accordingly, baseline blood pressure was elevated in the transgenic mice in response to acetylcholine in basilar artery. It states that the finding in the study presents a genetic evidence that endothelial PPARγ plays a critical role in protecting blood vessels.

The transcription factor PPARgamma is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPARgamma plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPARgamma (L/+). In L/+ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF(2alpha), and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/+ mice. Superoxide levels were elevated in cerebral arterioles from L/+ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/+ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPARgamma signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPARgamma has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPARgamma plays a critical role in protecting blood vessels. [ABSTRACT FROM AUTHOR]

• Complete loss of elastin is incompatible with life. • Elastin haploinsufficiency leads to adaptive changes in arterial wall development and function, including an increased number of thinner arterial elastic laminae and smooth muscle cell layers, large artery stiffness, alterations in small vessel reactivity, and systolic hypertension that are independent of renin levels. • Resistance artery changes, namely a significant change in endothelial cell function and hypercontractility to angiotensin II, point to pathway-specific alterations that contribute to the hypertensive phenotype in elastin haploinsufficiency. Supravalvular aortic stenosis (SVAS) is an autosomal dominant disease resulting from elastin (ELN) haploinsufficiency. Individuals with SVAS typically develop a thickened arterial media with an increased number of elastic lamellae and smooth muscle cell (SMC) layers and stenosis superior to the aortic valve. A mouse model of SVAS (Eln+/−) was generated that recapitulates many aspects of the human disease, including increased medial SMC layers and elastic lamellae, large artery stiffness, and hypertension. The vascular changes in these mice were thought to be responsible for the hypertension phenotype. However, a renin gene (Ren) duplication in the original 129/Sv genetic background and carried through numerous strain backcrosses raised the possibility of renin-mediated effects on blood pressure. To exclude excess renin activity as a disease modifier, we utilized the Cre-LoxP system to rederive Eln hemizygous mice on a pure C57BL/6 background (Sox2-Cre;Elnf/f). Here we show that Sox2-Cre;Eln+/f mice, with a single Ren1 gene and normal renin levels, phenocopy the original global knockout line. Characteristic traits include an increased number of elastic lamellae and SMC layers, stiff elastic arteries, and systolic hypertension with widened pulse pressure. Importantly, small resistance arteries of Sox2-Cre;Eln+/f mice exhibit a significant change in endothelial cell function and hypercontractility to angiotensin II, findings that point to pathway-specific alterations in resistance arteries that contribute to the hypertensive phenotype. These data confirm that the cardiovascular changes, particularly systolic hypertension, seen in Eln+/− mice are due to Eln hemizygosity rather than Ren duplication. [ABSTRACT FROM AUTHOR]

PPARγ is a ligand activated transcription factor. Dominant negative (DN) mutations in PPARγ have been reported in patients with type 2 diabetes and early onset hypertension. To explore the role of PPARγ in vascular smooth muscle cells (vSMC) in vivo, we generated transgenic (Tg) mice designed to interfere with PPARγ-dependent signaling by specifically targeting expression of a DN PPARγ (P467L) to SMC. Transgene expression in tissues containing SMC, including aorta was confirmed by RNase protection. Vascular function in aorta from Tg mice and non-Tg (NT) littermates was examined in vitro. Remarkably, aorta of Tg animals exhibited endothelial dysfunction and an impaired response to nitric oxide as evidenced by significantly lower relaxation to acetylcholine (ACh 10µM, 9±2%) and sodium nitroprusside (SNP 10µM, 44±4%) than aorta of NT (38±3% for ACh, 864-1% for SNP, P<0.001). Submaximal relaxation to the endothelial-independent vasodilator paperverine was slightly but significantly impaired. We are currently testing if the impaired response to NO is due to impaired cGMP-dependent signaling. Interestingly, aorta of Tg mice also contracted considerably more to endothelin-1 (ET-1 0.1 µM, 302±29 mg) than did aorta of NT (44±6 mg, P<0.001). This increase in contraction was inhibited by the ET-A receptor antagonist BQ-123. These data suggest that vSMC PPARγ plays a pivotal role in the regulation of vascular tone. [ABSTRACT FROM AUTHOR]

PPARγ is a ligand activated transcription factor, a target of anti-diabetic thiozolidinedione drugs, and exerts anti-oxidant and anti-inflammatory effects. However, its role in vascular biology is not well understood. We tested the hypothesis that PPARγ protects the vasculature under normal conditions. To address this question, we used heterozygous mice expressing a dominant negative (DN) mutation in PPARγ and measured reactivity in aorta and cerebral blood vessels. In aorta, responses to acetylcholine (ACh) were not affected, but there was a significant increase in contraction to serotonin, PGF2α, and endothelin-1. In the basilar artery in vitro, ACh produced dilation that was impaired in DN mice (e.g. 1 µM ACh, 61±9 vs 32±5%, DN vs control P<0.05). Using a cranial window in anesthetized mice, dilator responses of cerebral arterioles (∼30 µm in diameter) to ACh were selectively reduced in DN mice (29±3 vs 12±2%, 10 µM ACh, P<0.05) but were restored nearly to normal with Tempol, a scavenger of superoxide. Thus, interference with PPARγ signaling in mice produces endothelial dysfunction in cerebral arteries and arterioles via a mechanism involving oxidative stress. These findings indicate that PPARγ has functional effects in the cerebral circulation and provide the first direct evidence that PPARγ plays a critical role in protecting blood vessels. [ABSTRACT FROM AUTHOR]

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin- 4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B. [ABSTRACT FROM AUTHOR]