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Biomedical subjects

H Paul Ehrlich

Publications and source records attributed to H Paul Ehrlich.

At least 19 recordsLinked to original sources
PubMed2007

Rat mast cells communicate with fibroblasts via gap junction intercellular communications.

Usually mast cells (MCs) modulate other cellular activities through the release of their cytoplasmic granules. Recently, gap junctional intercellular communication (GJIC) between an established human MC cell line (HMC-1) co-cultured with human dermal fibroblasts in fibroblast populated collagen lattices (FPCLs), enhanced the rate and degree of FPCL contraction. However, HMC-1 cells were unable to generate GJIC with human neonatal fibroblasts in monolayer culture. Here freshly isolated rat peritoneal MCs are co-cultured with fibroblasts in collagen lattices and in monolayer culture in vitro and introduced into rat polyvinyl alcohol (PVA) sponge implants in vivo. Co-cultured MC-FPCL contracted faster and to a greater degree. Loading Calcein AM green fluorescent dye into red fluorescent Dil tagged MC generates MC-paratroopers. When MC-paratroopers form GJIC with fibroblasts, some green dye is passed into the fibroblast, while the MC-paratrooper retains both its red and green fluorescence. MC-paratroopers passed green fluorescent dye into both human and rat dermal fibroblasts in monolayer culture. In rats 7-day-old subcutaneous PVA sponge implants, which received an injection of MC-paratroopers, exhibited auto-fluorescent green fibroblasts, when harvested 24 h later. MC-paratroopers pretreated with a long-acting GJIC inhibitor prior to their introduction into PVA sponge implants, failed to pass dye into fibroblasts. It is proposed that GJIC between granulation tissue fibroblasts and MCs can modulate some aspects of wound repair and fibrosis.

Animals↗
PubMed2006

Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin.

In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum-free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler-treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen alpha1 (I) mRNA was minimally affected by endosulfan. Oleamide-treated 17-day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti-mouse-IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody-collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide-treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring.

Animals↗
PubMed2006

Tissue fibrosis and carcinogenesis: divergent or successive pathways dictate multiple molecular therapeutic targets for oligo decoy therapies.

The extracellular matrix (ECM) is composed of several families of macromolecular components: fibrous proteins such as collagens, type I collagen (COL1), type III collagen (COL3), fibronectin, elastin, and glycoconjugates such as proteoglycans and matrix glycoproteins. Their receptors on the cell membrane, most of which in the case of the ECM belong to the integrins, which are heterodimeric proteins composed of alpha and beta chains. COL1 is the major fibrous collagen of bone, tendon, and skin; while COL3 is the more pliable collagen of organs like liver. Focus will not only be given to the regulation of synthesis of several fibrogenic parameters but also modulation of their degradation during growth factor-induced tissue fibrosis and cancer development. Evidence will be provided that certain tissues, which undergo fibrosis, also become cancerous. Why does there exist a divergency between tissues, which undergo frank fibrosis as an endpoint, and those tissues that undergo fibrosis and subsequently are susceptible to carcinogenicity; resulting from the etiological factor(s) causing the initial injury? For example, why does a polyvinyl alcohol (PVA) sponge implant become encapsulated and filled with fibrous tissue then fibrosis tissue growth stops? Why does the subcutaneous injection of a fibrogenic growth factor cause a benign growth and incisional wounding results in fibrosis and ultimately scarring? There are many examples of tissues, which undergo fibrosis as a prerequisite to carcinogenesis. Is there a cause-effect relationship? If you block tissue fibrosis in these precancerous tissues, would you block cancer formation? What are the molecular targets for blocking fibrosis and ultimately carcinogenesis? How can oligo decoys may be used to attenuate carcinogenesis and which oligo decoys specifically attenuate fibrogenesis as a prelude to carcinogenesis? What are other molecular targets for oligo decoy therapy in carcinogenesis?

Animals↗
PubMed2005

Dynamic changes appearing in collagen fibers during intrinsic tendon repair.

Intrinsic healing of severed tendons shows a delay in a gain in breaking strength and the tendon becomes translucent. The cause of tendon translucence was investigated in suture-repaired rat Achilles tendon. The repair site with adjacent translucent tendon were evaluated histologically on day 10 by immunofluorescence and transmission electron microscopy. The healing tendon translucent region by hematoxylin-eosin staining had few inflammatory cells, polarized light birefringence showed thinner collagen fibers, immunofluorescence showed few myofibroblasts, and transmission electron microscopy revealed frayed, irregular thin collagen fibers. During embryogenesis, tendon fibers grow by the addition of discreet collagen fibril segment structures. The speculation is that collagen fibril segment structures are released from collagen fibers within the translucent tendon region for reuse during the regeneration of tendon collagen fibers during intrinsic tendon repair. Healing tendon translucence is related to a decrease in the diameter of collagen fibers by the release of collagen fibril segments within tendon bundles/fascicles.

Achilles Tendon↗
PubMed2004

A histological and anatomical profile of pacinian corpuscles from Dupuytren's contracture and the expression of nerve growth factor receptor.

The etiology of Dupuytren's disease is unknown. The causes of the fibroplastic response of nodules, fibrosis of cords, and prominence of pacinian corpuscles are not evident. Histological and immunohistology differences in pacinian corpuscles from the hands of five patients with Dupuytren's disease compared with 17 Dupuytren's-free patients are presented. Histological sections of pacinian corpuscle specimens were stained with hematoxylin and eosin and immunostained for nerve growth factor receptor. The length and width of intact pacinian corpuscles were measured, and the number of layers within each corpuscle was counted and recorded. Grossly, the pacinian corpuscles from Dupuytren's patients were larger and more numerous compared with those from unaffected patients. When measured microscopically, the pacinian corpuscles from Dupuytren's diseased fascia were significantly larger (2.0 x 1.1 mm) compared with controls (1.5 x 0.78 mm). The pacinian corpuscles from Dupuytren's-affected patients had significantly more layers (64 +/- 14) compared with those from control patients (40 +/- 9). Nerve growth factor receptor staining of pacinian corpuscles from patients affected with Dupuytren's disease showed greater intensity and more area stained compared with unaffected controls. It is suggested that nerve growth factor may be involved in the increased size of pacinian corpuscles in Dupuytren's-affected fascia. It is proposed that the cellular outgrowth from pacinian corpuscles may generate the cells that develop into Dupuytren's nodules.

Cell Line↗
PubMed2004

Understanding experimental biology of skin equivalent: from laboratory to clinical use in patients with burns and chronic wounds.

A major breakthrough in burn wound care was the early excision of the burn and its immediate coverage with a skin autograft. A search for a skin-graft substitute began to reduce the autografting-related trauma at the donor site. One entry was skin equivalence, which contains 3 components: (1) living fibroblasts, suspended in (2) a native collagen matrix, the surface of which is covered with (3) viable keratinocytes. The tissue-cultured dermal fibroblasts are derived from human foreskin. The fibroblasts are grown in cell culture dishes as a monolayer and are retrieved by limited trypsin digestion. The fibroblast suspension is mixed with serum-supplemented culture medium and native acid-soluble collagen. The entire mixture, called a dermal equivalent, is placed in a bacteriological Petri dish before transfer to a 37 degrees C incubator. The collagen rapidly polymerizes, trapping cells in the dermal equivalent. During the initial 4 hours, fibroblasts elongate and spread, causing a decrease in the thickness of the dermal equivalent. After 6 hours, the dermal equivalent undergoes a decrease in diameter as a consequence of the reorganization of the collagen. A freshly isolated suspension of human skin-derived keratinocytes is seeded on the surface of a several-day-old floating dermal equivalent. The keratinocytes proliferate, covering the surface of the dermal equivalent. The keratinocytes deposit basement membranes beneath them and undergo epidermal cell differentiation, leading to the formation of a basal layer beneath differentiated cell layers. Both cell populations retain viability and release cell factors that have a positive effect on wound closure. The placement of skin equivalence within a chronic wound may share structural attributes with a skin graft, but its function is to accelerate closure.

Animals↗
PubMed2004

Pulp nonfiction: microscopic anatomy of the digital pulp space.

The volar pad of the fingertip provides a very stable yet sensitive surface that gives the hand the ability to pinch and grasp. The focus of this study was to advance understanding of the anatomical features of the digital pulp space. The unusual features of the fingertip pulp space include prominent collagen fiber cords and a branching continuous fine vasculature. Prominent collagen fiber cords radiating out from beneath the epidermal basement membrane are like the cords of a parachute, which directly attach to the periosteum of the distal phalanx. Those collagen fiber cords are responsible for the firm attachment of the fingertip to the distal phalanx. There is a fine patent vasculature within the pulp space. Also contained in the capsule are numerous lobules of fat, which contribute to some elasticity of the fingertip. Principles of treatment for injuries or infections of the digital pulp should attempt to preserve this anatomical construct so that the firmness and vascular supply of the fingertip are maintained and not disrupted.

Collagen↗
PubMed2004

Integrin-linked kinase: a possible role in scar contracture.

Integrin-linked kinase (ILK) participates with beta1 integrin to mediate extracellular matrix interactions, such as extracellular matrix reorganization. Thus, ILK is hypothesized to influence wound contraction and scar contracture and, as such, would be a target molecule to manipulate pharmacologically in expediting wound contraction or possibly preventing scar contracture. The expression of ILK messenger ribonucleic acid, along with ILK-protein expression, was found in fibroblasts. The localization of ILK in human skin and rat granulation tissue was documented by immunohistology. ILK was present in human dermal fibroblasts, but was not found in human epidermal cells in skin. Cells were transfected with wild-type ILK or kinase-deficient ILK (E359K) and were assayed for collagen lattice contraction, migration, and myosin adenosine triphosphatase (ATPase) activity. Cells overexpressing E359K were poorer at collagen lattice contraction than control cells, whereas cells overexpressing wild-type ILK were equal to control cells at lattice contraction. ILK overexpression enhanced cell migration, but E359K overexpression did not affect cell migration. Neither ILK nor E359K overexpression altered myosin ATPase activity. Hence, ILK action within fibroblasts appears unrelated to myosin ATPase control of microfilament-generated forces. ILK appears to be a target molecule for pharmacologic manipulation to expedite wound contraction or to prevent scar contracture.

Animals↗
PubMed2004

Alpha V integrin prolongs collagenase production through Jun activation binding protein 1.

Robust expression of alphav integrin and matrix metalloproteinase 1 (MMP1) plays an important role in cancer metastasis and wound healing. A patient with an abnormal scar that appeared stretched and thinned out was found to have fibroblasts that overexpressed alphav integrin; therefore, a relationship between alphav integrin expression and MMP1 production was sought. A yeast 2 hybrid screen revealed alphav integrin interacts with jun activation binding domain-1 (JAB1). Mesenchymal-derived cells were transfected with the alphav integrin gene and incorporated into collagen lattices. Transfected cells maximally contracted collagen lattices beginning on day 5, whereas control transfected cells did not contract lattices. Late-phase collagen lattice contraction was inhibited by a pan-MMP inhibitor, BB4. Overexpression of alphav correlated with enhanced MMP1 transcription, as determined by a luciferase assay (P < or = 0.05). Diminution of JAB1 with JAB1 antisense abolished alphav integrin up-regulation of MMP1. We conclude alphav integrin signals through JAB1 to prolong MMP1 production and that this signaling pathway in fibroblasts may lead to abnormal scarring.

COP9 Signalosome Complex↗
PubMed2003

Systemic vanadate ingestion modulates rat tendon repair.

The chronic ingestion of vanadate prevents the appearance of myofibroblasts within granulation tissue of full excision wounds in rats, yet these wounds close at an optimal rate. Myofibroblasts are reported in the repair of transected tendons. Here we investigate tendon repair in the absence of myofibroblasts. Vanadate in saline drinking water was given to rats in the experimental group, while rats in the control group received saline alone. The Achilles tendon of the left leg of each rat was transected and suture repaired. On day 10, both repaired tendons and uninjured tendons from the right leg were harvested and processed for histology. By immunohistology the repaired tendons of control rats had myofibroblasts (fibroblasts with alpha smooth muscle actin positive stress fibers), while myofibroblasts were absent in healing tendons from vanadate-treated rats. By transmission electron microscopy and polarized light optics, repaired tendons of control rats demonstrated thin, loosely packed, immature collagen fiber bundles. Collagen fiber bundles from healing tendons of the vanadate-treated group were thicker, uniformly packed, and more mature. The chronic ingestion of vanadate promotes the more rapid organization of collagen fiber bundles of healing transected tendons in the absence of myofibroblasts.

Achilles Tendon↗
PubMed2003

Is the tendon embryogenesis process resurrected during tendon healing?

The process of embryonic tendon development, including the nature and purpose of collagen fibril segments, is reviewed. It is proposed that tendon fibrillogenesis of repair is related to the fibrillogenesis of tendon embryonic development. The assembly of collagen fibril segment units into longer fibers occurs on the surface of tendon fibroblasts in embryonic tendon development. The biochemist's view of tendon healing, whereby the spontaneous polymerization of tropocollagen monomers regenerates lost tendon collagen fibers, needs to be reconsidered. Furthermore, the importance of direct fibroblast involvement in collagen fiber reassembly during tendon healing needs to be studied in tendon intrinsic regenerative repair.

Animals↗
PubMed2003

Clinical efficacy and mechanism of bilayered living human skin equivalent (HSE) in treatment of diabetic foot ulcers.

UNLABELLED: Bilayered living human skin equivalent (HSE) consists of cultured keratinocytes residing on the surface of a fibroblast-populated collagen lattice. Although HSE is FDA-approved for treatment of diabetic foot and venous stasis ulcers, its clinical efficacy remains limited, because the molecular mechanisms underlying its therapeutic effect are not fully understood. It is, therefore, often applied mistakenly as a skin graft. In this report, we delineate a mechanism of HSE biological effect and consequent optimal clinical use in accelerating closure of diabetic foot ulcers. EXPERIMENTAL: HSE was grafted onto nude mice and the release of various growth factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Clinical: HSE was grafted onto 11 consecutive patients with diabetes who had 13 non-ischemic foot ulcers and healing was measured as time to 100% closure (e.g., no drainage and 100% epithelialized). EXPERIMENTAL: HSE cellular components were determined to express 15 different growth factors/cytokine genes known to promote wound healing. Histological evidence from the nude mice showed that the collagen component of HSE underwent remodeling within the first seven days of grafting. Clinical: All diabetic foot ulcers healed in 31.8 12.4 days. Local release of a unique combination of 15 growth factors expressed by HSE keratinocyte and fibroblast components generates closure of diabetic foot ulcers. HSE should be applied with the same surgical conditions for a skin graft (i.e., no cellulitis, no drainage, and negligible bacteria). We hypothesize that bilayered HSE generates its effect by way of the local synthesis and release of multiple growth factors in specific combination and concentration, which improves the impaired reparative process of chronic wounds.

Animals↗
PubMed2003

Healing of elderly patients with diabetic foot ulcers, venous stasis ulcers, and pressure ulcers.

Although elderly patients have physiologic impairments in wound healing, their wounds should be expected to heal with the same frequency of closure as those in younger populations, albeit at a slower rate. However, compared to the general population, the elderly population has a higher incidence of chronic wounds: diabetic foot ulcers, pressure ulcers, and venous stasis ulcers. Experimental and clinical data indicate physiologically impaired healing is characterized by decreased angiogenesis and synthesis of critical growth factors. Further, compared to younger populations, the elderly have a higher rate of mortality associated with specific morbidities, such as sepsis and acute respiratory distress. As these morbidities may develop directly from the wound, early intervention is mandated. In this report, 40 consecutive elderly patients (65-102 years old) with chronic wounds were analyzed. All patients were provided the same treatment protocol and healing was defined as 100% epithelization and no drainage. Despite the wounds presenting in a nonhealing and/or infected state, 73% of these chronic wounds in elderly patients healed. This suggests that elderly patients with diabetic foot ulcers, pressure ulcers, and venous stasis ulcers close their wounds at a similar frequency as younger patients. Therefore, early intervention and comprehensive treatment that includes safe topical therapies, in addition to growth factors and cellular therapy used for chronic wounds, ensure these patients will be spared the morbidities of pain, amputation, osteomyelitis, and even death. We hypothesize that if all elderly patients with chronic wounds are provided early treatment, morbidities (e.g., amputation, sepsis, pain) and associated costs will decrease.

Age Factors↗
PubMed2002

Overexpression of integrin alphav promotes human osteosarcoma cell populated collagen lattice contraction and cell migration.

Cells attach and interact with the extracellular matrix (ECM) through heterodimeric alphabeta integrin receptors. Specifically, the promiscuous alphavbeta3 integrin and the alpha2beta1 integrin receptors engage numerous matrix components to influence cell adhesion, cell motility, and matrix organization. However, the role of alphav integrin mediating cell-collagen interactions is not clear. In the in vitro cell populated collagen lattice (PCL), a model of cell-matrix interaction, integrin receptors play a role in lattice contraction. To elucidate alphav integrins' effects on cell-collagen interactions, human osteosarcoma (HOS) cells were transfected with alphav integrin (alphav-pcDNA 3.1+). Control HOS cells were transfected with pcDNA 3.1+ vector alone. HOS-alphav cell PCLs contracted to a greater degree than control HOS cell PCLs (P < or = 0.0001). RT-PCR revealed that HOS-alphav cells express both beta1 and beta3 integrins, indicating that alphav has the potential to form a partnership with either beta1 or beta3 integrin. The alphavbeta3 specific inhibitory antibody LM609 significantly retarded HOS-alphav cell PCL contraction (P < or = 0.001), suggesting that alphavbeta3 promotes enhanced HOS-alphav cell PCL contraction. When plated on plastic, control HOS cells show greater elongation compared to HOS-alphav cells. In addition, HOS-alphav cells migrated faster and to a greater degree than control HOS cells (P < or = 0.0001). The possibility that enhanced HOS-alphav cell migration and HOS-alphav cell PCL contraction was caused by increased myosin ATPase activity was examined. HOS-alphav cells showed less myosin ATPase activity than control HOS cells, by an ATP cell contraction bioassay. The enhancement of HOS-alphav cell migration and lattice contraction appears unrelated to increased myosin ATPase activity.

Antibodies, Monoclonal↗
PubMed2002

Topical platelet-derived growth factor in patients enhances wound closure in the absence of wound contraction.

Recombinant human platelet-derived growth factor (PDGF) is reported to promote wound closure in problem wounds. The mechanism of PDGF enhancement of wound closure is not clear. Does PDGF enhance wound contraction, or re-epithelialization, or both? In four patients undergoing elective surgery, a full excision 5 cm(2) punch wound was made behind each ear. The left post-articular wound received daily PDGF in a gel and each right post-articular wound received placebo-gel daily. The placebo-treated wounds closed in 19.8 days, while the PDGF-treated wounds closed significantly faster, in 15.6 days, p=0.002. At Day 20, all healed wounds were processed for histology. PDGF-treated wounds showed granulation tissue beneath an uninterrupted epidermis. A fine birefringence pattern, consistent with granulation tissue, was found by polarized light microscopy. The control closed-wounds had a smaller area of granulation tissue under an intact epidermis and polarized light microscopy showed mostly normal dermis. The presence of intact dermis within the closed-wound site is the hallmark of wound contraction. Topical PDGF limits the role of wound contraction in wound closure. Control wounds healed by wound contraction, while PDGF-treated wounds close by re-epithelialization and filling in with scar.

Administration, Topical↗
PubMed2002

Topical platelet-derived growth factor enhances wound closure in the absence of wound contraction: an experimental and clinical study.

The effects of platelet-derived growth factor (PDGF) on wound healing in animal and human models were investigated. Four 1-cm2 wounds were made on the dorsum of 3 rats. A 0.5-cm punch wound was made behind each ear of 4 patients. Half the wounds were treated daily with vehicle, controls, and the rest were treated with PDGF. Treated wounds closed faster than the controls (animals: 16 +/- 3.2 days vs. 17.8 +/- 2.17 days; p < 0.05) and (patients: 16 +/- 0.67 days vs. 19.5 +/- 0.33 days; p < 0.05). Biopsies were taken at day 20 for polarized light-Sirius red histological analysis. The granulation tissue of PDGF-treated wounds showed fine collagen fibers with weak birefringence, characteristic of immature granulation tissue, deposited throughout the healed wound site. Such a pattern indicates wound closure by reepithelialization and filling in with scar. Control wound biopsies showed a small area of immature granulation tissue surrounded by intact dermal thick collagen fibers with strong birefringence. Such a pattern indicates wound closure by wound contraction. This shows that PDGF enhances wound closure by reepithelialization and the prevention of wound contraction.

Animals↗
PubMed2002

Dupuytren's disease: physiologic changes in nodule and cord fibroblasts through aging in vitro.

The pathogenesis of the fibrotic disease Dupuytren's contracture remains unclear. The disease process includes two structurally distinct fibrotic elements, the nodule and the cord. It has been proposed that as the disease progresses, nodules develop into cords. To corroborate that hypothesis, the authors took advantage of cultured fibroblast differences found between gap junction intercellular communication and fibroblast-populated collagen lattice contraction. Paired fibroblast cell lines of nodules and cords derived from four patients with Dupuytren's disease were maintained in culture for at least eight passages. The presence of gap junction intercellular communication in nodule- and cord-derived fibroblasts was documented and reported as a coupling index. The contraction of free-floating nodule- or cord-derived collagen lattices was also documented and reported. Early passage (passage 4) cord-derived fibroblasts showed a significant increase in coupling index compared with passage 4 nodule-derived fibroblasts (4.0 +/- 0.4 versus 2.5 +/- 0.3, respectively), where p < or = 0.01. However, late passage (passage 8) nodule- and cord-derived fibroblasts were equivalent in their coupling index (4.1 +/- 0.4 versus 4.4 +/- 0.4, respectively). Early passage nodule-derived fibroblast-populated collagen lattices contracted by 64 percent, whereas late passage nodule-derived lattices showed less contraction, at only 40 percent. Early and late passage cord-derived lattices contracted 46 and 37 percent, respectively. All nodule- and cord-derived cell lines were statistically equivalent at lattice contraction by passage 8. These in vitro studies support the hypothesis that fibroblasts derived from Dupuytren's contracture nodules change their phenotype after undergoing repeated cell passage, acquiring a cord-like fibroblast phenotype. Dupuytren's nodules represent the early, active form of fibrosis in which cells are more proliferative, better at fibroblast-populated collagen lattice contraction, and display less gap junction intercellular communication. The speculation is that alterations in gap junction intercellular communication may be involved in the progression of Dupuytren's nodules to cords as the disease progresses.

Cell Communication↗