Multi-organ phenotypes in mice lacking latent TGFß binding protein 2 (LTBP2).

BACKGROUND: Latent TGFß binding protein-2 (LTBP2) is a fibrillin 1 binding component of the microfibril. LTBP2 is the only LTBP protein that does not bind any isoforms of TGFß, although it may interfere with the function of other LTBPs or interact with other signaling pathways. RESULTS: Here, we investigate mice lacking Ltbp2 (Ltbp2-/- ) and identify multiple phenotypes that impact bodyweight and fat mass, and affect bone and skin development. The alterations in skin and bone development are particularly noteworthy since the strength of these tissues is differentially affected by loss of Ltbp2. Interestingly, some tissues that express high levels of Ltbp2, such as the aorta and lung, do not have a developmental or homeostatic phenotype. CONCLUSIONS: Analysis of these mice show that LTBP2 has complex effects on development through direct effects on the extracellular matrix (ECM) or on signaling pathways that are known to regulate the ECM.

A new mouse model of elastin haploinsufficiency highlights the importance of elastin to vascular development and blood pressure regulation.

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.

Loss of Angiotensin II Type 2 Receptor Improves Blood Pressure in Elastin Insufficiency.

There is ample evidence supporting a role for angiotensin II type 2 receptor (AT2R) in counterbalancing the effects of angiotensin II (ang II) through the angiotensin II type 1 receptor by promoting vasodilation and having anti-inflammatory effects. Elastin insufficiency in both humans and mice results in large artery stiffness and systolic hypertension. Unexpectedly, mesenteric arteries from elastin insufficient (Eln +/-) mice were shown to have significant vasoconstriction to AT2R agonism in vitro suggesting that AT2R may have vasoconstrictor effects in elastin insufficiency. Given the potential promise for the use of AT2R agonists clinically, the goal of this study was to determine whether AT2R has vasoconstrictive effects in elastin insufficiency in vivo. To avoid off-target effects of agonists and antagonists, mice lacking AT2R (Agtr2 -/Y ) were bred to Eln +/- mice and cardiovascular parameters were assessed in wild-type (WT), Agtr2 -/Y , Eln +/-, and Agtr2 -/Y ;Eln +/- littermates. As previously published, Agtr2 -/Y mice were normotensive at baseline and had no large artery stiffness, while Eln +/- mice exhibited systolic hypertension and large artery stiffness. Loss of AT2R in Eln +/- mice did not affect large artery stiffness or arterial structure but resulted in significant reduction of both systolic and diastolic blood pressure. These data support a potential vasocontractile role for AT2R in elastin insufficiency. Careful consideration and investigation are necessary to determine the patient population that might benefit from the use of AT2R agonists.

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

OBJECTIVE: Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models. CONCLUSIONS: These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Encephalitic Alphaviruses Exploit Caveola-Mediated Transcytosis at the Blood-Brain Barrier for Central Nervous System Entry.

Venezuelan and western equine encephalitis viruses (VEEV and WEEV, respectively) invade the central nervous system (CNS) early during infection, via neuronal and hematogenous routes. While viral replication mediates host shutoff, including expression of type I interferons (IFN), few studies have addressed how alphaviruses gain access to the CNS during established infection or the mechanisms of viral crossing at the blood-brain barrier (BBB). Here, we show that hematogenous dissemination of VEEV and WEEV into the CNS occurs via caveolin-1 (Cav-1)-mediated transcytosis (Cav-MT) across an intact BBB, which is impeded by IFN and inhibitors of RhoA GTPase. Use of reporter and nonreplicative strains also demonstrates that IFN signaling mediates viral restriction within cells comprising the neurovascular unit (NVU), differentially rendering brain endothelial cells, pericytes, and astrocytes permissive to viral replication. Transmission and immunoelectron microscopy revealed early events in virus internalization and Cav-1 association within brain endothelial cells. Cav-1-deficient mice exhibit diminished CNS VEEV and WEEV titers during early infection, whereas viral burdens in peripheral tissues remained unchanged. Our findings show that alphaviruses exploit Cav-MT to enter the CNS and that IFN differentially restricts this process at the BBB.IMPORTANCE VEEV, WEEV, and eastern equine encephalitis virus (EEEV) are emerging infectious diseases in the Americas, and they have caused several major outbreaks in the human and horse population during the past few decades. Shortly after infection, these viruses can infect the CNS, resulting in severe long-term neurological deficits or death. Neuroinvasion has been associated with virus entry into the CNS directly from the bloodstream; however, the underlying molecular mechanisms have remained largely unknown. Here, we demonstrate that following peripheral infection alphavirus augments vesicular formation/trafficking at the BBB and utilizes Cav-MT to cross an intact BBB, a process regulated by activators of Rho GTPases within brain endothelium. In vivo examination of early viral entry in Cav-1-deficient mice revealed significantly lower viral burdens in the brain than in similarly infected wild-type animals. These studies identify a potentially targetable pathway to limit neuroinvasion by alphaviruses.

Heterogeneous Cellular Contributions to Elastic Laminae Formation in Arterial Wall Development.

RATIONALE: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.

Nonlinear optical imaging of extracellular matrix proteins.

Over the last 2 decades, nonlinear imaging methods such as multiharmonic imaging microscopy (MHIM) have become powerful approaches for the label-free visualization of biological structures. Multiharmonic signals are generated when an intense electromagnetic field propagates through a sample that either has a specific molecular orientation or exhibits certain physical properties. It can provide complementary morphological information when integrated with other nonlinear optical imaging techniques such as two-photon excitation (TPE). Here, we present the necessary methodology to implement an integrated approach for multiharmonic and TPE imaging of the mouse aorta using a commercial two-photon microscope. This approach illustrates how to differentiate the microstructure of the mouse aorta that are due to collagen fibrils and elastic laminae under 820 and 1230nm excitation. Our method also demonstrates how to perform multiharmonic generation by reflectance of the forwardly propagating emission signal. The ability to visualize biological samples without additional genetically targeted or chemical stains makes MHIM well suited for studying the morphology of the mouse aorta and has the potential to be applied to other collagen and elastin-rich tissues.

Inducible Expression of Claudin-1 in Glomerular Podocytes Generates Aberrant Tight Junctions and Proteinuria through Slit Diaphragm Destabilization.

The tight junction (TJ) has a key role in regulating paracellular permeability to water and solutes in the kidney. However, the functional role of the TJ in the glomerular podocyte is unclear. In diabetic nephropathy, the gene expression of claudins, in particular claudin-1, is markedly upregulated in the podocyte, accompanied by a tighter filtration slit and the appearance of TJ-like structures between the foot processes. However, there is no definitive evidence to show slit diaphragm (SD) to TJ transition in vivo Here, we report the generation of a claudin-1 transgenic mouse model with doxycycline-inducible transgene expression specifically in the glomerular podocyte. We found that induction of claudin-1 gene expression in mature podocytes caused profound proteinuria, and with deep-etching freeze-fracture electron microscopy, we resolved the ultrastructural change in the claudin-1-induced SD-TJ transition. Notably, immunolabeling of kidney proteins revealed that claudin-1 induction destabilized the SD protein complex in podocytes, with significantly reduced expression and altered localization of nephrin and podocin proteins. Mechanistically, claudin-1 interacted with both nephrin and podocin through cis- and trans-associations in cultured cells. Furthermore, the rat puromycin aminonucleoside nephrosis model, previously suspected of undergoing SD-TJ transition, exhibited upregulated expression levels of claudin-1 mRNA and protein in podocytes. Together, our data attest to the novel concept that claudins and the TJ have essential roles in podocyte pathophysiology and that claudin interactions with SD components may facilitate SD-TJ transition that appears to be common to many nephrotic conditions.

Ultrastructural properties of ciliary zonule microfibrils.

Conventional electron microscopy and rotary shadowing techniques have provided conflicting interpretations of microfibril ultrastructure. To address this issue, we have used quick-freeze deep-etch (QFDE) microscopy to obtain 3-dimensional surface views of microfibrils that have not been fixed, dehydrated, or stained with heavy metals. By this approach, microfibrils appear as tightly packed rows of bead-like subunits that do not display the interbead filamentous links seen by other methods. At regular 50-nm intervals along the microfibril length, a larger bead is often recognized which tends to be aligned with those from adjacent microfibrils when the microfibrils are in bundles. This evidence of organized lateral associations of microfibrils is supported by the observation of small filaments that span between the adjacent microfibrils. When QFDE microscopy was used to examine microfibrils exposed to sonication, partially dissociated microfibrils with the more typical "beads on a string" appearance were observed. Beads are also seen alone, as monomers, often with an array of small thread-like filaments extending from the bead in a "crab-like" manner. Our results suggest that the beads on a string appearance of sonicated microfibrils may result from a partial loss of protein components from the interbead domains, thus leading to exposure of a filamentous substructure. It is possible, therefore, that this phenomenon might also contribute to the beads on a string appearance of microfibrils seen using other electron microscopy techniques.