Combined genetic-pharmacologic inactivation of tightly linked ADAMTS proteases in temporally specific windows uncovers distinct roles for versican proteolysis and glypican-6 in cardiac development.

Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6-/- hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.

SARS-CoV-2 Brain Regional Detection, Histopathology, Gene Expression, and Immunomodulatory Changes in Decedents with COVID-19.

Brains of 42 COVID-19 decedents and 107 non-COVID-19 controls were studied. RT-PCR screening of 16 regions from 20 COVID-19 autopsies found SARS-CoV-2 E gene viral sequences in 7 regions (2.5% of 320 samples), concentrated in 4/20 subjects (20%). Additional screening of olfactory bulb (OB), amygdala (AMY) and entorhinal area for E, N1, N2, RNA-dependent RNA polymerase, and S gene sequences detected one or more of these in OB in 8/21 subjects (38%). It is uncertain whether these RNA sequences represent viable virus. Significant histopathology was limited to 2/42 cases (4.8%), one with a large acute cerebral infarct and one with hemorrhagic encephalitis. Case-control RNAseq in OB and AMY found more than 5000 and 700 differentially expressed genes, respectively, unrelated to RT-PCR results; these involved immune response, neuronal constituents, and olfactory/taste receptor genes. Olfactory marker protein-1 reduction indicated COVID-19-related loss of OB olfactory mucosa afferents. Iba-1-immunoreactive microglia had reduced area fractions in cerebellar cortex and AMY, and cytokine arrays showed generalized downregulation in AMY and upregulation in blood serum in COVID-19 cases. Although OB is a major brain portal for SARS-CoV-2, COVID-19 brain changes are more likely due to blood-borne immune mediators and trans-synaptic gene expression changes arising from OB deafferentation.

Hemispheric Asymmetry and Atypical Lobar Progression of Alzheimer-Type Tauopathy.

The spread of neurofibrillary tau pathology in Alzheimer disease (AD) mostly follows a stereotypical pattern of topographical progression but atypical patterns associated with interhemispheric asymmetry have been described. Because histopathological studies that used bilateral sampling are limited, this study aimed to assess interhemispheric tau pathology differences and the presence of topographically atypical cortical spreading patterns. Immunohistochemical staining for detection of tau pathology was performed in 23 regions of interest in 57 autopsy cases comparing bilateral cortical regions and hemispheres. Frequent mild (82% of cases) and occasional moderate (32%) interhemispheric density discrepancies were observed, whereas marked discrepancies were uncommon (7%) and restricted to occipital regions. Left and right hemispheric tau pathology dominance was observed with similar frequencies, except in Braak Stage VI that favored a left dominance. Interhemispheric Braak stage differences were observed in 16% of cases and were more frequent in advanced (IV-VI) versus early (I-III) stages. One atypical lobar topographical pattern in which occipital tau pathology density exceeded frontal lobe scores was identified in 4 cases favoring a left dominant asymmetry. We speculate that asymmetry and atypical topographical progression patterns may be associated with atypical AD clinical presentations and progression characteristics, which should be tested by comprehensive clinicopathological correlations.

Olfactory Bulb and Amygdala Gene Expression Changes in Subjects Dying with COVID-19.

In this study we conducted RNA sequencing on two brain regions (olfactory bulb and amygdala) from subjects who died from COVID-19 or who died of other causes. We found several-fold more transcriptional changes in the olfactory bulb than in the amygdala, consistent with our own work and that of others indicating that the olfactory bulb may be the initial and most common brain region infected. To some extent our results converge with pseudotime analysis towards common processes shared between the brain regions, possibly induced by the systemic immune reaction following SARS-CoV-2 infection. Changes in amygdala emphasized upregulation of interferon-related neuroinflammation genes, as well as downregulation of synaptic and other neuronal genes, and may represent the substrate of reported acute and subacute COVID-19 neurological effects. Additionally, and only in olfactory bulb, we observed an increase in angiogenesis and platelet activation genes, possibly associated with microvascular damages induced by neuroinflammation. Through coexpression analysis we identified two key genes (CAMK2B for the synaptic neuronal network and COL1A2 for the angiogenesis/platelet network) that might be interesting potential targets to reverse the effects induced by SARS-CoV-2 infection. Finally, in olfactory bulb we detected an upregulation of olfactory and taste genes, possibly as a compensatory response to functional deafferentation caused by viral entry into primary olfactory sensory neurons. In conclusion, we were able to identify transcriptional profiles and key genes involved in neuroinflammation, neuronal reaction and olfaction induced by direct CNS infection and/or the systemic immune response to SARS-CoV-2 infection.

Vagus Nerve and Stomach Synucleinopathy in Parkinson's Disease, Incidental Lewy Body Disease, and Normal Elderly Subjects: Evidence Against the "Body-First" Hypothesis.

BACKGROUND: Braak and others have proposed that Lewy-type α-synucleinopathy in Parkinson's disease (PD) may arise from an exogenous pathogen that passes across the gastric mucosa and then is retrogradely transported up the vagus nerve to the medulla. OBJECTIVE: We tested this hypothesis by immunohistochemically staining, with a method specific for p-serine 129 α-synuclein (pSyn), stomach and vagus nerve tissue from an autopsy series of 111 normal elderly subjects, 33 with incidental Lewy body disease (ILBD) and 53 with PD. METHODS: Vagus nerve samples were taken adjacent to the carotid artery in the neck. Stomach samples were taken from the gastric body, midway along the greater curvature. Formalin-fixed paraffin-embedded sections were immunohistochemically stained for pSyn, shown to be highly specific and sensitive for α-synuclein pathology. RESULTS: Median disease duration for the PD group was 13 years. In the vagus nerve none of the 111 normal subjects had pSyn in the vagus, while 12/26 ILBD (46%) and 32/36 PD (89%) subjects were pSyn-positive. In the stomach none of the 102 normal subjects had pSyn while 5/30 (17%) ILBD and 42/52 (81%) of PD subjects were pSyn-positive. CONCLUSION: As there was no pSyn in the vagus nerve or stomach of subjects without brain pSyn, these results support initiation of pSyn in the brain. The presence of pSyn in the vagus nerve and stomach of a subset of ILBD cases indicates that synucleinopathy within the peripheral nervous system may occur, within a subset of individuals, at preclinical stages of Lewy body disease.

Acute Brain Ischemia, Infarction and Hemorrhage in Subjects Dying with or Without Autopsy-Proven Acute Pneumonia.

Stroke is one of the most serious complications of Covid-19 disease but it is still unclear whether stroke is more common with Covid-19 pneumonia as compared to non-Covid-19 pneumonia. We investigated the concurrence rate of autopsy-confirmed acute brain ischemia, acute brain infarction and acute brain hemorrhage with autopsy-proven acute non-Covid pneumonia in consecutive autopsies in the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study of normal aging and neurodegenerative diseases. Of 691 subjects with a mean age of 83.4 years, acute pneumonia was histopathologically diagnosed in 343 (49.6%); the concurrence rates for histopathologically-confirmed acute ischemia, acute infarction or subacute infarction was 14% and did not differ between pneumonia and non-pneumonia groups while the rates of acute brain hemorrhage were 1.4% and 2.0% of those with or without acute pneumonia, respectively. In comparison, in reviews of Covid-19 publications, reported clinically-determined rates of acute brain infarction range from 0.5% to 20% while rates of acute brain hemorrhage range from 0.13% to 2%. In reviews of Covid-19 autopsy studies, concurrence rates for both acute brain infarction and acute brain hemorrhage average about 10%. Covid-19 pneumonia and non-Covid-19 pneumonia may have similar risks tor concurrent acute brain infarction and acute brain hemorrhage when pneumonia is severe enough to cause death. Additionally, acute brain ischemia, infarction or hemorrhage may not be more common in subjects dying of acute pneumonia than in subjects dying without acute pneumonia.

Mapping of SARS-CoV-2 Brain Invasion and Histopathology in COVID-19 Disease.

The coronavirus SARS-CoV-2 (SCV2) causes acute respiratory distress, termed COVID-19 disease, with substantial morbidity and mortality. As SCV2 is related to previously-studied coronaviruses that have been shown to have the capability for brain invasion, it seems likely that SCV2 may be able to do so as well. To date, although there have been many clinical and autopsy-based reports that describe a broad range of SCV2-associated neurological conditions, it is unclear what fraction of these have been due to direct CNS invasion versus indirect effects caused by systemic reactions to critical illness. Still critically lacking is a comprehensive tissue-based survey of the CNS presence and specific neuropathology of SCV2 in humans. We conducted an extensive neuroanatomical survey of RT-PCR-detected SCV2 in 16 brain regions from 20 subjects who died of COVID-19 disease. Targeted areas were those with cranial nerve nuclei, including the olfactory bulb, medullary dorsal motor nucleus of the vagus nerve and the pontine trigeminal nerve nuclei, as well as areas possibly exposed to hematogenous entry, including the choroid plexus, leptomeninges, median eminence of the hypothalamus and area postrema of the medulla. Subjects ranged in age from 38 to 97 (mean 77) with 9 females and 11 males. Most subjects had typical age-related neuropathological findings. Two subjects had severe neuropathology, one with a large acute cerebral infarction and one with hemorrhagic encephalitis, that was unequivocally related to their COVID-19 disease while most of the 18 other subjects had non-specific histopathology including focal ß-amyloid precursor protein white matter immunoreactivity and sparse perivascular mononuclear cell cuffing. Four subjects (20%) had SCV2 RNA in one or more brain regions including the olfactory bulb, amygdala, entorhinal area, temporal and frontal neocortex, dorsal medulla and leptomeninges. The subject with encephalitis was SCV2-positive in a histopathologically-affected area, the entorhinal cortex, while the subject with the large acute cerebral infarct was SCV2-negative in all brain regions. Like other human coronaviruses, SCV2 can inflict acute neuropathology in susceptible patients. Much remains to be understood, including what viral and host factors influence SCV2 brain invasion and whether it is cleared from the brain subsequent to the acute illness.

Increased Risk of Autopsy-Proven Pneumonia with Sex, Season and Neurodegenerative Disease.

There has been a markedly renewed interest in factors associated with pneumonia, a leading cause of death worldwide, due to its frequent concurrence with pandemics of influenza and Covid-19 disease. Reported predisposing factors to both bacterial pneumonia and pandemic viral lower respiratory infections are wintertime occurrence, older age, obesity, pre-existing cardiopulmonary conditions and diabetes. Also implicated are age-related neurodegenerative diseases that cause parkinsonism and dementia. We investigated the prevalence of autopsy-proven pneumonia in the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND), a longitudinal clinicopathological study, between the years 2006 and 2019 and before the beginning of the Covid-19 pandemic. Of 691 subjects dying at advanced ages (mean 83.4), pneumonia was diagnosed postmortem in 343 (49.6%). There were 185 subjects without dementia or parkinsonism while clinicopathological diagnoses for the other subjects included 319 with Alzheimer's disease dementia, 127 with idiopathic Parkinson's disease, 72 with dementia with Lewy bodies, 49 with progressive supranuclear palsy and 78 with vascular dementia. Subjects with one or more of these neurodegenerative diseases all had higher pneumonia rates, ranging between 50 and 61%, as compared to those without dementia or parkinsonism (40%). In multivariable logistic regression models, male sex and a non-summer death both had independent contributions (ORs of 1.67 and 1.53) towards the presence of pneumonia at autopsy while the absence of parkinsonism or dementia was a significant negative predictor of pneumonia (OR 0.54). Male sex, dementia and parkinsonism may also be risk factors for Covid-19 pneumonia. The apolipoprotein E4 allele, as well as obesity, chronic obstructive pulmonary disease, diabetes, hypertension, congestive heart failure, cardiomegaly and cigarette smoking history, were not significantly associated with pneumonia, in contradistinction to what has been reported for Covid-19 disease.

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation.

Focal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.

Stromal Versican Regulates Tumor Growth by Promoting Angiogenesis.

The proteoglycan versican is implicated in growth and metastases of several cancers. Here we investigated a potential contribution of stromal versican to tumor growth and angiogenesis. We initially determined versican expression by several cancer cell lines. Among these, MDA-MB231 and B16F10 had none to minimal expression in contrast to Lewis lung carcinoma (LLC). Notably, tumors arising from these cell lines had higher versican levels than the cell lines themselves suggesting a contribution from the host-derived tumor stroma. In LLC-derived tumors, both the tumor and stroma expressed versican at high levels. Thus, tumor stroma can make a significant contribution to tumor versican content. Versican localized preferentially to the vicinity of tumor vasculature and macrophages in the tumor. However, an ADAMTS protease-generated versican fragment uniquely localized to vascular endothelium. To specifically determine the impact of host/stroma-derived versican we therefore compared growth of tumors from B16F10 cells, which produced littleversican, in Vcan hdf/+ mice and wild-type littermates. Tumors in Vcan hdf/+ mice had reduced growth with a lower capillary density and accumulation of capillaries at the tumor periphery. These findings illustrate the variability of tumor cell line expression of versican, and demonstrate that versican is consistently contributed by the stromal tissue, where it contributes to tumor angiogenesis.

ADAMTS9-Mediated Extracellular Matrix Dynamics Regulates Umbilical Cord Vascular Smooth Muscle Differentiation and Rotation.

Despite the significance for fetal nourishment in mammals, mechanisms of umbilical cord vascular growth remain poorly understood. Here, the secreted metalloprotease ADAMTS9 is shown to be necessary for murine umbilical cord vascular development. Restricting it to the cell surface using a gene trap allele, Adamts9(Gt), impaired umbilical vessel elongation and radial growth via reduced versican proteolysis and accumulation of extracellular matrix (ECM). Both Adamts9(Gt) and conditional Adamts9 deletion revealed that ADAMTS9 produced by mesenchymal cells acted non-autonomously to regulate smooth muscle cell (SMC) proliferation, differentiation, and orthogonal reorientation during growth of the umbilical vasculature. In Adamts9(Gt/Gt), we observed interference with PDGFRß signaling via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which regulates cytoskeletal dynamics during SMC rotation. In addition, we observed disrupted Shh signaling and perturbed orientation of the mesenchymal primary cilium. Thus, ECM dynamics is a major influence on umbilical vascular SMC fate, with ADAMTS9 acting as its principal mediator.

Determinants of versican-V1 proteoglycan processing by the metalloproteinase ADAMTS5.

Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) ß domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGß domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond.

A new Adamts9 conditional mouse allele identifies its non-redundant role in interdigital web regression.

ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5-8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ-line deletion produced from the floxed allele by Cre-lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1-Cre mice. Unlike other ADAMTS single knockouts, limb-specific Adamts9 deletion resulted in soft-tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non-redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9.

Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis.

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFß signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

Altered versican cleavage in ADAMTS5 deficient mice; a novel etiology of myxomatous valve disease.

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for 'remodeling' the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5(-/-) mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5(-/-) valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5(-/-) valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5(-/-) mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation.

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFß3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.

Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies.

Here, we demonstrate that ADAMTS9, a highly conserved versican-degrading protease, is required for correct cardiovascular development and adult homeostasis. Analysis of Adamts9(+/LacZ) adult mice revealed anomalies in the aortic wall, valvulosinus and valve leaflets. Abnormal myocardial projections and 'spongy' myocardium consistent with non-compaction of the left ventricle were also found in Adamts9(+/LacZ) mice. During development, Adamts9 was expressed in derivatives of the Secondary Heart Field, vascular smooth muscle cells in the arterial wall, mesenchymal cells of the valves, and non-myocardial cells of the ventricles, but expression also continued in the adult heart and ascending aorta. Thus, the adult cardiovascular anomalies found in Adamts9(+/LacZ) hearts could result from subtle developmental alterations in extracellular matrix remodeling or defects in adult homeostasis. The valvular and aortic anomalies of Adamts9(+/LacZ) hearts were associated with accumulation of versican and a decrease in cleaved versican relative to WT littermates. These data suggest a potentially important role for ADAMTS9 cleavage of versican, or other, as yet undefined substrates in development and allostasis of cardiovascular extracellular matrix. In addition, these studies identify ADAMTS9 as a potential candidate gene for congenital cardiac anomalies. Mouse models of ADAMTS9 deficiency may be useful to study myxomatous valve degeneration.

ADAMTS9 is a cell-autonomously acting, anti-angiogenic metalloprotease expressed by microvascular endothelial cells.

The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.

ADAMTS metalloproteases generate active versican fragments that regulate interdigital web regression.

We show that combinatorial mouse alleles for the secreted metalloproteases Adamts5, Adamts20 (bt), and Adamts9 result in fully penetrant soft-tissue syndactyly. Interdigital webs in Adamts5(-/-);bt/bt mice had reduced apoptosis and decreased cleavage of the proteoglycan versican; however, the BMP-FGF axis, which regulates interdigital apoptosis was unaffected. BMP4 induced apoptosis, but without concomitant versican proteolysis. Haploinsufficiency of either Vcan or Fbln1, a cofactor for versican processing by ADAMTS5, led to highly penetrant syndactyly in bt mice, suggesting that cleaved versican was essential for web regression. The local application of an aminoterminal versican fragment corresponding to ADAMTS-processed versican, induced cell death in Adamts5(-/-);bt/bt webs. Thus, ADAMTS proteases cooperatively maintain versican proteolysis above a required threshold to create a permissive environment for apoptosis. The data highlight the developmental significance of proteolytic action on the ECM, not only as a clearance mechanism, but also as a means to generate bioactive versican fragments.