Kelly A. Hogan

A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells

We characterize a sonic hedgehog (Shh) signaling domain restricted to the adventitial layer of artery wall that supports resident Sca1-positive vascular progenitor cells (AdvSca1). Using patched-1 (Ptc1lacZ) and patched-2 (Ptc2lacZ) reporter mice, adventitial Shh signaling activity was first detected at embryonic day (E) 15.5, reached the highest levels between postnatal day 1 (P1) and P10, was diminished in adult vessels, and colocalized with a circumferential ring of Shh protein deposited between the media and adventitia. In Shh−/− mice, AdvSca1 cells normally found at the aortic root were either absent or greatly diminished in number. Using a Wnt1-cre lineage marker that identifies cells of neural crest origin, we found that neither the adventitia nor AdvSca1 cells were labeled in arteries composed of neural crest-derived smooth muscle cells (SMCs). Although AdvSca1 cells do not express SMC marker proteins in vivo, they do express transcription factors thought to be required for SMC differentiation, including serum response factor (SRF) and myocardin family members, and readily differentiate to SMC-like cells in vitro. However, AdvSca1 cells also express potent repressors of SRF-dependent transcription, including Klf4, Msx1, and FoxO4, which may be critical for maintenance of the SMC progenitor phenotype of AdvSca1 cells in vivo. We conclude that a restricted domain of Shh signaling is localized to the arterial adventitia and may play important roles in maintenance of resident vascular SMC progenitor cells in the artery wall.

The neural tube patterns vessels developmentally using the VEGF signaling pathway.

Embryonic blood vessels form in a reproducible pattern that interfaces with other embryonic structures and tissues, but the sources and identities of signals that pattern vessels are not well characterized. We hypothesized that the neural tube provides vascular patterning signal(s) that direct formation of the perineural vascular plexus (PNVP) that encompasses the neural tube at mid-gestation. Both surgically placed ectopic neural tubes and ectopic neural tubes engineered genetically were able to recruit a vascular plexus, showing that the neural tube is the source of a vascular patterning signal. In mouse-quail chimeras with the graft separated from the neural tube by a buffer of host cells, graft-derived vascular cells contributed to the PNVP, indicating that the neural tube signal(s) can act at a distance. Murine neural tube vascular endothelial growth factor A (VEGFA) expression was temporally and spatially correlated with PNVP formation, suggesting it is a component of the neural tube signal. A collagen explant model was developed in which presomitic mesoderm explants formed a vascular plexus in the presence of added VEGFA. Co-cultures between presomitic mesoderm and neural tube also supported vascular plexus formation, indicating that the neural tube could replace the requirement for VEGFA. Moreover, a combination of pharmacological and genetic perturbations showed that VEGFA signaling through FLK1 is a required component of the neural tube vascular patterning signal. Thus, the neural tube is the first structure identified as a midline signaling center for embryonic vascular pattern formation in higher vertebrates, and VEGFA is a necessary component of the neural tube vascular patterning signal. These data suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning.

Getting Under the Hood: How and for Whom Does Increasing Course Structure Work?

The authors explore the transferability of an active-learning intervention and expand upon the original studies by 1) disaggregating student populations to identify for whom the intervention works best and 2) exploring possible proximate mechanisms (changes in student behaviors and perceptions) that could mediate the observed increase in achievement.

Blood Vessel Patterning at the Embryonic Midline

The reproducible pattern of blood vessels formed in vertebrate embryos has been described extensively, but only recently have we obtained the genetic and molecular tools to address the mechanisms underlying these processes. This review describes our current knowledge regarding vascular patterning around the vertebrate midline and presents data derived from frogs, zebrafish, avians, and mice. The embryonic structures implicated in midline vascular patterning, the hypochord, endoderm, notochord, and neural tube, are discussed. Moreover, several molecular signaling pathways implicated in vascular patterning, VEGF, Tie/tek, Notch, Eph/ephrin, and Semaphorin, are described. Data showing that VEGF is critical to patterning the dorsal aorta in frogs and zebrafish, and to patterning the vascular plexus that forms around the neural tube in amniotes, is presented. A more complete knowledge of vascular patterning is likely to come from the next generation of experiments using ever more sophisticated tools, and these results promise to directly impact on clinically important issues such as forming new vessels in the human body and/or in bioreactors.

Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment γ316–322, part of the fibrin‐specific region*

Summary. The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12‐carboxy‐terminal residues of the γ‐chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the γ‐chain segments γ318–320 and γ408–411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to γΔ408‐411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: γΔ319‐320, γ318Asp→Ala and γ320Asp→Ala. Furthermore, antibodies raised against the sequences γ308–322 and γ316–333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment γ316–322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin‐specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

Fibrinogen Otsu I:A γ Asn319,Asp320 deletion dysfibrinogen identified in an asymptomatic pregnant woman

Fibrinogen Otsu I:A γ Asn319,Asp320 deletion dysfibrinogen identified in an asymptomatic pregnant woman -

The Formation of β Fibrin Requires a Functional a Site

Abstract: We used recombinant fibrinogens in which the a site is disrupted to examine β‐fibrin formation in the absence of a functional a site. Our variants have only b sites available, and they showed no evidence of fibrin polymer formation after cleavage of FpB with venzyme. We conclude that B‐b interactions are not strong enough to induce clot formation. Our studies do not rule out the involvement of b in the formation of β‐fibrin, yet they do provide evidence that a is likely to be essential in the formation of β‐fibrin.

Neonatal bleeding and decreased plasma fibrinogen levels in mice modeled after the dysfibrinogen Vlissingen/Frankfurt IV

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Polymerization Site a Function Dependence on Structural Integrity of Its Nearby Calcium Binding Site

Abstract: To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: γD364A in the a site and γD318A, γD320A, and γD318 +γD320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS‐PAGE analysis of the products showed that γD364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected γD318A, γD320A, or γD318 +γD320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.

Synthesis of a Mouse Model of the Dysfibrinogen Vlissingen/Frankfurt IV

Abstract: The dysfibrinogen Vlissingen/Frankfurt IV is characterized as a deletion of Asn319 and Asp320 from the C‐terminus of the γ‐chain of fibrinogen. This dysfibrinogen, which was identified in several family members that are all heterozygous for the in‐frame 6‐bp deletion, is associated with both venous and arterial thrombosis. Here, we describe the generation of a murine model of the V/F IV dysfibrinogen using gene targeting of mouse γ‐chain DNA. Preliminary analysis shows that the human and mouse variant fibrinogens are similar: analogous to the human V/F IV protein, the D1 fragment of the variant mouse fibrinogen is partially protected from digestion in the presence of calcium or Gly‐Pro‐Arg‐Pro. These heterozygous mice provide the first opportunity to examine the association of thrombophilia and dysfibrinogenemia in a controlled genetic background.