Dario Bizzotto

Vascular Smooth Muscle Emilin-1 Is a Regulator of Arteriolar Myogenic Response and Blood Pressure

Objective—Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-&bgr; processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. Methods and Results—This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1−/− background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1−/− mice. We also explored the effect of treatment with anti-TGF-&bgr; antibodies on the hypertensive phenotype of Emilin1−/− mice, finding that neutralization of TGF-&bgr; in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-&bgr; activity. Conclusion—Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-&bgr;.

EMILIN-3, Peculiar Member of Elastin Microfibril Interface-located Protein (EMILIN) Family, Has Distinct Expression Pattern, Forms Oligomeric Assemblies, and Serves as Transforming Growth Factor β (TGF-β) Antagonist

Background: EMILIN-3 is the least characterized member of the EMILIN/Multimerin family. Results: EMILIN-3 forms homotrimers and higher order oligomers, binds heparin, has a dynamic expression during development and a restricted distribution in adult tissues, and serves as a pro-TGF-β antagonist. Conclusion: The structure and expression of EMILIN-3 are different from other EMILINs/Multimerins. Significance: EMILIN-3, a TGF-β antagonist, is likely to be an important regulator during development of several tissues. EMILIN-3 is a glycoprotein of the extracellular matrix belonging to a family that contains a characteristic N-terminal cysteine-rich EMI domain. Currently, EMILIN-3 is the least characterized member of the elastin microfibril interface-located protein (EMILIN)/Multimerin family. Using RNA, immunohistochemical, and protein chemistry approaches, we carried out a detailed characterization of the expression and biochemical properties of EMILIN-3 in mouse. During embryonic and postnatal development, EMILIN-3 showed a peculiar and dynamic pattern of gene expression and protein distribution. EMILIN-3 mRNA was first detected at E8.5–E9.5 in the tail bud and in the primitive gut, and at later stages it became abundant in the developing gonads and osteogenic mesenchyme. Interestingly and in contrast to other EMILIN/Multimerin genes, EMILIN-3 was not found in the cardiovascular system. Despite the absence of the globular C1q domain, immunoprecipitation and Western blot analyses demonstrated that EMILIN-3 forms disulfide-bonded homotrimers and higher order oligomers. Circular dichroism spectroscopy indicated that the most C-terminal part of EMILIN-3 has a substantial α-helical content and forms coiled coil structures involved in EMILIN-3 homo-oligomerization. Transfection experiments with recombinant constructs showed that the EMI domain contributes to the higher order self-assembly but was dispensable for homotrimer formation. EMILIN-3 was found to bind heparin with high affinity, a property mediated by the EMI domain, thus revealing a new function for this domain that may contribute to the interaction of EMILIN-3 with other extracellular matrix and/or cell surface molecules. Finally, in vitro experiments showed that EMILIN-3 is able to function as an extracellular regulator of the activity of TGF-β ligands.

An enhancer required for transcription of the Col6a1 gene in muscle connective tissue is induced by signals released from muscle cells

Collagen VI is a survival factor for skeletal muscle produced by endomysial cells and localized in connective tissue around muscle fibers. Mutations of its genes (COL6A1, COL6A2 and COL6A3) cause two muscular disorders, Bethlem myopathy and Ullrich disease. Expression of Collagen VI is highly dynamic during development, suggesting that developmental and homeostatic cues of the muscle microenvironment are relevant to confine its expression in this tissue. In face of the large body of work highlighting the relevance for human diseases of the adhesion of muscle cells with their surrounding extracellular matrix, remarkably little is known on how myogenic cells control gene expression in the connective tissue cells that produce such matrix. By expressing promoter-lacZ constructs in transgenic mice, we identify a Col6a1 gene enhancer region that is necessary for activation of transcription in connective tissue cells associated with skeletal muscle. By means of a lacZ transgenic mouse line crossed in metD/D mutant background, in which muscles of limb buds fail to form, we provide evidence that the presence of cells of the myogenic lineage is needed for enhancer activation in mesenchymal cells. Accordingly, lack of myogenic cells in limb buds of metD/D mice reduces Collagen VI deposition in connective tissue. The Col6a1 enhancer characterized here is conserved in mammals and may be relevant in some cases of heritable diseases of Collagen VI.

Targeting Interleukin-1β Protects from Aortic Aneurysms Induced by Disrupted Transforming Growth Factor β Signaling

SUMMARY Aortic aneurysms are life‐threatening conditions with effective treatments mainly limited to emergency surgery or trans‐arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor &bgr; (TGF‐&bgr;) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)‐specific inactivation of Smad4, an intracellular transducer of TGF‐&bgr;. The results revealed that Smad4 inhibition activated interleukin‐1&bgr; (IL‐1&bgr;) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C‐C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC‐specific Smad4 deletion in Il1r1‐ or Ccr2‐null mice resulted in milder aortic pathology. A chronic treatment with anti‐IL‐1&bgr; antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF‐&bgr; signaling, such as those driven by SMAD4 mutations. HIGHLIGHTSSMC‐specific inducible deletion of Smad4 in adult mice provokes aortic aneurysmsThe resulting disrupted TGF‐&bgr; signaling activates IL‐1&bgr; in SMCs as a danger signalIL‐1&bgr; induces CCL2 to recruit an innate immune response harmful to the aortic wallsTargeting IL‐1&bgr; and CCL2 protects mice with disrupted TGF‐&bgr; in SMCs from aneurysms TGF‐&bgr; signaling has an unquestionable but still controversial role in the pathogenesis of aortic aneurysm. Da Ros et al. demonstrate that disruption of TGF‐&bgr; signaling in SMCs activates an autocrine IL‐1&bgr; pathway that acts as a danger signal to recruit innate immune cells in the adventitia through CCL2.

The ablation of the matricellular protein EMILIN2 causes defective vascularization due to impaired EGFR-dependent IL-8 production affecting tumor growth

EMILIN2 is an extracellular matrix constituent playing an important role in angiogenesis; however, the underlying mechanism is unknown. Here we show that EMILIN2 promotes angiogenesis by directly binding epidermal growth factor receptor (EGFR), which enhances interleukin-8 (IL-8) production. In turn, IL-8 stimulates the proliferation and migration of vascular endothelial cells. Emilin2 null mice were generated and exhibited delayed retinal vascular development, which was rescued by the administration of the IL-8 murine ortholog MIP-2. Next, we assessed tumor growth and tumor-associated angiogenesis in these mice. Tumor cell growth in Emilin2 null mice was impaired as well as the expression of MIP-2. The vascular density of the tumors developed in Emilin2 null mice was prejudiced and vessels perfusion, as well as response to chemotherapy, decreased. Accordingly, human tumors expressing high levels of EMILIN2 were more responsive to chemotherapy. These results point at EMILIN2 as a key microenvironmental cue affecting vessel formation and unveil the possibility to develop new prognostic tools to predict chemotherapy efficacy.

Collagen VI null mice as a model for early onset muscle decline in aging

Collagen VI is an extracellular matrix (ECM) protein playing a key role in skeletal muscles and whose deficiency leads to connective tissue diseases in humans and in animal models. However, most studies have been focused on skeletal muscle features. We performed an extensive proteomic profiling in two skeletal muscles (diaphragm and gastrocnemius) of wild-type and collagen VI null (Col6a1−/−) mice at different ages, from 6- (adult) to 12- (aged) month-old to 24 (old) month-old. While in wild-type animals the number of proteins and the level of modification occurring during aging were comparable in the two analyzed muscles, Col6a1−/− mice displayed a number of muscle-type specific variations. In particular, gastrocnemius displayed a limited number of dysregulated proteins in adult mice, while in aged muscles the modifications were more pronounced in terms of number and level. In diaphragm, the differences displayed by 6-month-old Col6a1−/− mice were more pronounced compared to wild-type mice and persisted at 12 months of age. In adult Col6a1−/− mice, the major variations were found in the enzymes belonging to the glycolytic pathway and the tricarboxylic acid (TCA) cycle, as well as in autophagy-related proteins. When compared to wild-type animals Col6a1−/− mice displayed a general metabolic rewiring which was particularly prominent the diaphragm at 6 months of age. Comparison of the proteomic features and the molecular analysis of metabolic and autophagic pathways in adult and aged Col6a1−/− diaphragm indicated that the effects of aging, culminating in lipotoxicity and autophagic impairment, were already present at 6 months of age. Conversely, the effects of aging in Col6a1−/− gastrocnemius were similar but delayed becoming apparent at 12 months of age. A similar metabolic rewiring and autophagic impairment was found in the diaphragm of 24-month-old wild-type mice, confirming that fatty acid synthase (FASN) increment and decreased microtubule-associated proteins 1A/1B light chain 3B (LC3B) lipidation are hallmarks of the aging process. Altogether these data indicate that the diaphragm of Col6a1−/− animal model can be considered as a model of early skeletal muscle aging.

EMILIN3, an extracellular matrix molecule with restricted distribution in skin

EMILIN3 is an extracellular matrix glycoprotein that displays a dynamic and restricted expression pattern in connective tissues during post‐natal life. In this study, we report the characterization of EMILIN3 deposition in the skin. In addition, to unravel the functions of this protein in skin homeostasis, we generated Emilin3 null mice and provide evidence that EMILIN3 is dispensable for hair follicle growth and maintenance throughout adult life.