Susan J. Braunhut

Degradation Products of Extracellular Matrix Affect Cell Migration and Proliferation

Biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles. The objective of the present study was to determine if degradation of ECM scaffold materials in vitro by methods that are biochemically and physiologically relevant can yield products that possess chemotactic and/or mitogenic activities for fully differentiated mammalian endothelial cells and undifferentiated multipotential progenitor cells. ECM harvested from porcine urinary bladder was degraded enzymatically with pepsin/hydrochloric acid or papain. The ECM degradation products were tested for chemoattractant properties utilizing either 48-well chemotaxis filter migration microchambers or fluorescence-based filter migration assays, and were tested for mitogenic properties in cell proliferation assays. Results showed that ECM degradation products possessed chemotactic and mitogenic activities for multipotential progenitor cells and that the same degradation products inhibited both chemotaxis and proliferation of differentiated endothelial cells. These findings support the concept that degradation products of ECM bioscaffolds are important modulators of the recruitment and proliferation of appropriate cell types during the process of ECM scaffold remodeling.

Epimorphic regeneration approach to tissue replacement in adult mammals

Urodeles and fetal mammals are capable of impressive epimorphic regeneration in a variety of tissues, whereas the typical default response to injury in adult mammals consists of inflammation and scar tissue formation. One component of epimorphic regeneration is the recruitment of resident progenitor and stem cells to a site of injury. Bioactive molecules resulting from degradation of extracellular matrix (ECM) have been shown to recruit a variety of progenitor and stem cells in vitro in adult mammals. The ability to recruit multipotential cells to the site of injury by in vivo administration of chemotactic ECM degradation products in a mammalian model of digit amputation was investigated in the present study. Adult, 6- to 8-week-old C57/BL6 mice were subjected to midsecond phalanx amputation of the third digit of the right hind foot and either treated with chemotactic ECM degradation products or left untreated. At 14 days after amputation, mice treated with ECM degradation products showed an accumulation of heterogeneous cells that expressed markers of multipotency, including Sox2, Sca1, and Rex1 (Zfp42). Cells isolated from the site of amputation were capable of differentiation along neuroectodermal and mesodermal lineages, whereas cells isolated from control mice were capable of differentiation along only mesodermal lineages. The present findings demonstrate the recruitment of endogenous stem cells to a site of injury, and/or their generation/proliferation therein, in response to ECM degradation products.

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo.

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.

A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations☆

The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Deltaf and DeltaR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11-15 microM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape. We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action.

Radiation-induced apoptosis in microvascular endothelial cells.

The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary endothelial cells (ECs) to ionizing radiation and bFGF. Apoptosis was assessed by identifying changes in nuclear morphology, recording cell detachment rates and by detecting internucleosomal DNA fragmentation. Withdrawal of bFGF alone induces apoptosis in these monolayers. The magnitude of this apoptotic response depends upon the duration of bFGF withdrawal. Irradiation (2-10 Gy) induces apoptosis in a dose-dependent manner. Radiation-induced apoptosis occurs in a discrete wave 6-10 h after irradiation, and radiation-induced apoptosis is enhanced in cultures that are simultaneously deprived of bFGF. For example, 6 h after 10 Gy, 44.3% (s.e. 6.3%) of cells in the monolayer simultaneously deprived of bFGF exhibit apoptotic morphology compared with 19.8% (s.e. 3.8%) in the presence of bFGF. These studies show that either bFGF withdrawal or ionizing radiation can induce apoptosis in confluent monolayers of capillary endothelial cells and that radiation-induced apoptosis can be modified by the presence of bFGF.

Quartz Crystal Microbalance Study of Endothelial Cell Number Dependent Differences in Initial Adhesion and Steady-State Behavior: Evidence for Cell-Cell Cooperativity in Initial Adhesion and Spreading

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Δf and ΔR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Δf and ΔR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Δf and ΔR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Δf and ΔR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Δf and ΔR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Δf and ΔR versus trypsinized, attached EC number plot 1 h post‐seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post‐seeding exhibits a hyperbolic curve shape. This number dependence suggests cell‐cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post‐addition, steady‐state Δf and ΔR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady‐state 24 h Δf and ΔR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy‐dissipative structure. This is consistent with the time‐dependent elaboration of surface contacts in anchorage‐dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.

Oxidative stress-induced apoptosis of endothelial cells.

Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O2*-. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 microM menadione resulted in an apoptotic rate of approximately 5% in capillary EC cultures compared with approximately 45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 microM), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 microM for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.

Copper and copper-binding protein in fibrolamellar liver cell carcinoma.

Analysis of tissue from a recent case of fibrolamellar liver cell carcinoma by several staining and spectrophotometrc methods demonstrated elevated copper and copper‐binding protein (CBP) in malignant hepatocytes. Production of CBP has not previously been described for this or any other type of hepatocellular carcinoma. Identification of CBP in liver cell carcinoma adds further evidence that this protein is a normal synthetic product of liver cells which may reappear in chronic cholestasis or hepatic malignancy. The mechanism of disturbed copper homeostasis in this case is uncertain.

Modulation of endothelial cell shape and growth by retinoids

Physiologic concentrations of retinol (1 X 10(-6) M) caused capillary and aortic endothelial cells (EC) to undergo a morphologic change, characterized by a rounder cell body, increased refractility at cell edges, and longer cytoplasmic processes distributed in a bipolar fashion. Computer image analysis of retinoid-treated EC revealed that both retinoic acid and retinol affected cellular area. Twenty-four hours following retinoic acid treatment, EC occupied a greater area than control (P less than 0.03) or retinol-treated EC (P less than 0.02). By Day 7, however, retinoic acid-treated EC occupied equivalent cellular areas as compared to control cells (P = 0.8). In contrast, by Day 7, retinol-treated EC occupied a smaller cellular area than control (P less than 0.002) or retinoic acid-treated EC (P less than 0.001). Proliferation studies revealed that within the first 72 hr of retinol treatment, basal EC growth was inhibited by 33% and the cells exhibited a lowered responsiveness to basic fibroblast growth factor (bFGF). In contrast, EC treated with retinoic acid and pericytes treated with each of the retinoids were not inhibited. The inhibitory effect of the 72 hr retinol treatment was reversible. Following 3 days exposure to retinol, EC given fresh media without retinoid underwent a population doubling in a subsequent 3-day period. However, in the continued presence of retinol, EC were 100% growth-inhibited. After a 3-day pretreatment with retinol, with or without continued retinol treatment, EC were refractile to the mitogenic action of bFGF in a subsequent 3-day period. These results demonstrate that retinol inhibits the basal and growth factor-stimulated growth of EC and causes a significant shape alteration of EC, supporting our hypothesis that vitamin A may be one of the signals that modify the growth and phenotype of EC.

Novel Synthetic SOD/Catalase Mimetics Can Mitigate Capillary Endothelial Cell Apoptosis Caused by Ionizing Radiation

Abstract Numerous in vitro and in vivo studies have shown that the endothelial cells of the microvasculature of the lung and kidney are damaged by exposure to ionizing radiation, and this sustained endothelial cell injury is involved in the early and late radiation effects observed in these tissues. It is well accepted that ionizing radiation causes the generation of reactive oxygen species during exposure that results in damage to DNA and cellular organelles. It is more controversial, however, whether additional biochemical events or long-lived radicals occur and persist postirradiation that amplify and initiate new forms of cellular damage. Two families of Eukarion (EUK) compounds have been synthesized that possess superoxide dismutase (SOD), catalase and peroxidase activities. The Mn porphyrins are available orally whereas the salen Mn complexes are administered by injection. In the present study we have examined the ability of these SOD/catalase mimetics to prevent apoptosis of endothelial cells when administered 1 h postirradiation (mitigation). A range of salen Mn complex (EUK-189 and EUK-207) and Mn porphyrins (EUK-418, -423, -425, -450, -451, -452, -453) were used to treat endothelial cells 1 h after the cells received 2–20 Gy ionizing radiation in vitro. Two lead compounds, EUK-207 at a dose of 30 µM and EUK-451 at a dose of 10 µM, exhibited low toxicity and mitigated radiation-induced apoptosis. Future animal studies will test whether these compounds protect when administered after radiation exposure as would be done after a radiological accident or a terrorism event.