Ira M. Herman

Bone morphogenetic protein expression in human atherosclerotic lesions.

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.

Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture

The transport of mRNAs into developing dendrites and axons may be a basic mechanism to localize cytoskeletal proteins to growth cones and influence microfilament organization. Using isoform-specific antibodies and probes for in situ hybridization, we observed distinct localization patterns for β- and γ-actin within cultured cerebrocortical neurons. β-Actin protein was highly enriched within growth cones and filopodia, in contrast to γ-actin protein, which was distributed uniformly throughout the cell. β-Actin protein also was shown to be peripherally localized after transfection of β-actin cDNA bearing an epitope tag. β-Actin mRNAs were localized more frequently to neuronal processes and growth cones, unlike γ-actin mRNAs, which were restricted to the cell body. The rapid localization of β-actin mRNA, but not γ-actin mRNA, into processes and growth cones could be induced by dibutyryl cAMP treatment. Using high-resolution in situ hybridization and image-processing methods, we showed that the distribution of β-actin mRNA within growth cones was statistically nonrandom and demonstrated an association with microtubules. β-Actin mRNAs were detected within minor neurites, axonal processes, and growth cones in the form of spatially distinct granules that colocalized with translational components. Ultrastructural analysis revealed polyribosomes within growth cones that colocalized with cytoskeletal filaments. The transport of β-actin mRNA into developing neurites may be a sequence-specific mechanism to synthesize cytoskeletal proteins directly within processes and growth cones and would provide an additional means to deliver cytoskeletal proteins over long distances.

Dynamic reciprocity in the wound microenvironment.

Here, we define dynamic reciprocity (DR) as an ongoing, bidirectional interaction among cells and their surrounding microenvironment. In this review, we posit that DR is especially meaningful during wound healing as the DR‐driven biochemical, biophysical, and cellular responses to injury play pivotal roles in regulating tissue regenerative responses. Such cell–extracellular matrix interactions not only guide and regulate cellular morphology, but also cellular differentiation, migration, proliferation, and survival during tissue development, including, e.g., embryogenesis, angiogenesis, as well as during pathologic processes including cancer, diabetes, hypertension, and chronic wound healing. Herein, we examine DR within the wound microenvironment while considering specific examples across acute and chronic wound healing. This review also considers how a number of hypotheses that attempt to explain chronic wound pathophysiology may be understood within the DR framework. The implications of applying the principles of DR to optimize wound care practice and future development of innovative wound healing therapeutics are also briefly considered.

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin Iβ) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

The pericyte: Cellular regulator of microvascular blood flow

The vascular system - through its development, response to injury, and remodeling during disease - constitutes one of the key organ systems sustaining normal human physiology; conversely, its dysregulation also underlies multiple pathophysiologic processes. Regulation of vascular endothelial cell function requires the integration of complex signals via multiple cell types, including arterial smooth muscle, capillary and post-capillary pericytes, and other perivascular cells such as glial and immune cells. Here, we focus on the pericyte and its roles in microvascular remodeling, reviewing current concepts in microvascular pathophysiology and offering new insights into the specific roles that pericyte-dependent signaling pathways may play in modulating endothelial growth and microvascular tone during pathologic angiogenesis and essential hypertension.

Inhibition of Calpain Blocks Platelet Secretion, Aggregation, and Spreading

Previous studies have indicated that the Ca2+-dependent protease, calpain, is activated in platelets within 30–60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpains specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposis fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced α-granule secretion (IC50 = 20 μm) during the first 30 s of activation, thrombin-induced platelet aggregation (IC50 = 50 μm), and platelet spreading on glass surfaces (IC50 = 34 μm). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3–10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

Microvascular Modifications in Diabetic Retinopathy

Patients struggling with diabetes are at elevated risks for several sight-threatening diseases, including proliferative diabetic retinopathy (DR). DR manifests in two stages: first, the retinal microvasculature is compromised and capillary degeneration occurs; subsequently, an over-compensatory angiogenic response is initiated. Early changes in the retinal microcirculation include disruptions in blood flow, thickening of basement membrane, eventual loss of mural cells, and the genesis of acellular capillaries. Endothelial apoptosis and capillary dropout lead to a hypoxic inner retina, alterations in growth factors, and upregulation of inflammatory mediators. With disease progression, pathologic angiogenesis generates abnormal preretinal microvessels. Current therapies, which include panretinal photocoagulation and vitrectomy, have remained unaltered for several decades. With several exciting preclinical advances, emergent technologies and innovative cellular targets may offer newfound hope for developing “next-generation” interventional or preventive clinical approaches that will significantly advance current standards of care and clinical outcomes.

Tumor angiogenesis: insights and innovations.

Angiogenesis is a vital process resulting in the formation of new blood vessels. It is normally a highly regulated process that occurs during human development, reproduction, and wound repair. However, angiogenesis can also become a fundamental pathogenic process found in cancer and several other diseases. To date, the inhibition of angiogenesis has been researched at both the bench and the bedside. While several studies have found moderate improvements when treating with angiogenesis inhibitors, greater success is being seen when the inhibition of angiogenesis is combined with other traditional forms of available therapy. This review summarizes several important angiogenic factors, examines new research and ongoing clinical trials for such factors, and attempts to explain how this new knowledge may be applied in the fight against cancer and other angiogenic-related diseases.

beta-Actin is confined to structures having high capacity of remodelling in developing and adult rat cerebellum.

Neurons undergo complex morphological changes during differentiation and in cases of plasticity. A major determinant of cell morphology is the actin cytoskeleton, which in neurons is comprised of two actin isoforms, non‐muscle γ‐ and β‐actin. To better understand their respective roles during differentiation and plasticity, their cellular and subcellular localization was examined in developing and adult cerebellar cortex. It was observed that γ‐actin is expressed at a constant level throughout development, while the level of β‐actin expression rapidly decreases with age. At the light microscopic level, γ‐actin staining is ubiquitous and the only developmental change observed is a relative reduction of its concentration in cell bodies and white matter. In contrast, β‐actin staining almost completely disappears from the cytoplasm of cell bodies, primary dendrites and axons. In young cerebellar cultures, γ‐actin is found in the cell body, neurites and growth cones, while β‐actin is mainly found in growth cones, as previously reported in other primary neuronal culture systems [Kaech et al. (1997), J. Neuroscience, 17, 9565–9572; Bassell et al. (1998), J. Neuroscience, 18, 251–265]. Electron microscopy of post‐embedding immunogold‐labelled tissue confirms the widespread distribution of γ‐actin, and also reveals an increased concentration of γ‐actin in dendritic spines in the adult. During development, β‐actin accumulation is observed in actively growing structures, e.g. growth cones, filopodia, cell bodies and axonal tracts. In the adult cerebellar cortex, β‐actin is preferentially found in dendritic spines, structures which are known to retain their capacity for morphological modifications in the adult brain. This differential subcellular localization and developmental regulation of the two actin isoforms point to their different roles in neurons.

Pericytes Derived from Adipose-Derived Stem Cells Protect against Retinal Vasculopathy

Background Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. Methodology/Principal Findings We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-β1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). Conclusions/Significance ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-β1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.